Cis di-ahl modified controls for glycated hemoglobin s-a1c derived from healthy blood cells

ABSTRACT

The invention is composition comprising red blood cells in an aqueous suspension medium and one or more cis di-ahls; wherein more than 6 percent by weight of the hemoglobin in the red blood cells is S-Alc glycated hemoglobin. In another embodiment, the invention is a method comprising contacting red blood cells in a suspension medium having a concentration of S-Alc glycated hemoglobin of greater than 6 percent by weight of the hemoglobin in the red blood cells with a sufficient amount of one or more cis di-ahls such that the concentration of S-Alc glycated hemoglobin in resulting composition as measured by high pressure liquid chromatography, immunoassay and boronate affinity methods is consistent.

CLAIM OF PRIORITY

This application claims priority from provisional application Ser. No.61/414,621 filed Nov. 17, 2010; provisional application Ser. No.61/414,631 filed Nov. 17, 2010 and provisional application Ser. No.61/414,633 filed Nov. 17, 2010 all incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates generally to compositions derived from healthyred blood cells containing S-Alc glycated hemoglobin and cis di-ahls,methods of preparing these compositions, controls containing thesecompositions wherein the controls are useful to calibrate analyticalinstruments used to determine the amount of glycated hemoglobincontained in blood samples. The present invention relates to controlcompositions and the manufacture and use of the same pursuant to whichthe red blood cells of the control (e.g., red blood cells obtained fromwhole blood of a donor who need not be diabetic) are processed in amanner for realizing simulated red blood cells, in relevantcharacteristics, of a diabetic person so that when analyzed by aninstrument capable of detecting such characteristics, the instrumentdetects such characteristics. Even more specifically the simulated redblood cells are such that they include glycosolated hemoblobin, which iscontained in a sealed cell membrane, and which is a result of Alcsynthesis that occurs within the membrane.

BACKGROUND OF THE INVENTION

In the diagnosis, treatment and management of patients with diabetes,sensitive instruments are employed for detecting one or more measurablecharacteristics of a patient blood sample. In a clinical laboratoryenvironment there is a need to ensure that such instruments areperforming properly. The use of control compositions having knowncharacteristics is an accepted way to assure proper instrumentperformance. Though it is possible to use human whole blood as acontrol, it is not desirable due to stability considerations as well asavailability. For example, in this context there would be a need for aready supply of fresh diabetic whole blood. Another accepted approach isto synthesize a control composition that simulates relevant detectablecharacteristics measured by an instrument. An approach to one suchsynthetic control composition is illustrated in U.S. Pat. No. 7,361,513(Ryan). Though such an approach yields useful control compositions,there remains a need for other such compositions. For example, there hasbeen a longstanding need for a control that does not require diabeticblood as a starting material, that employs Alc synthesis entirely withina cell membrane, or any combination thereof. There is also a need forachieving a control that has in-situ synthesized Alc contained within amembrane in an amount of a nature so that it is detectable as such by anumber of different instruments which may employ differing detectionstrategies. Hemoglobin (Hb) is a respiratory molecule found in red bloodcells. It is responsible for transporting oxygen from the lungs to bodycells and for transporting carbon dioxide from body cells to the lungs.Hemoglobin may be modified by the free glucose present in human plasmato form glycated hemoglobin (GHB). Hemoglobin Alc (Hb Alc, also referredto as Alc), constituting approximately 80 percent of all glycated Hb, isgenerated by the spontaneous reaction of glucose with the N-terminalamino group of the Hb A beta chain. The Hb Alc and the total glycated Hbvalues have a high degree of correlation, and either value may be used,for example in the management of treating diabetes. Formation of Hb Alcis slow but irreversible, and the blood level depends on both the lifespan of the red blood cells (average 120 days) and the blood glucoseconcentration. Therefore, Hb Alc represents the time-averaged bloodglucose values over the preceding 2 to 3 months, and is not subject towide fluctuations observed in blood glucose values. With respect todiabetes management, studies have shown that quality of life improveswith decreasing levels of Hb Alc, and measurements every 3 to 6 monthsare recommended.

The determination of total hemoglobin is indicative of theoxygen-carrying capacity of whole blood. The numerous methods anddevices for the determination of hemoglobin include both directanalysis, i.e., analysis without prior modification of the hemoglobin,and indirect analysis. It is important to accurately determine the totalhemoglobin in the Hb Alc assay, because Alc is often reported as afraction of the total hemoglobin. Multiple Hb Alc assay methodologieshave been developed since late 1970s. One of the standard methods formeasuring Hb Alc uses ionic-exchange high performance liquidchromatography (HPLC), which separates and analyzes Hb Alc and otherminor Hb components from unmodified hemoglobin (Hb A0) based upon theirdifferences in chemical charges. A second methodology for detection ofHb Alc is designed by immunoinhibition turbidimetric techniques. The HbAlc assay in immunoassay includes an antibody-antigen reaction and afollowing turbidity measurement. The third methodology is boronateaffinity chromatography, which utilizes a gel matrix containingimmobilized boronic acid to capture the cis-diol group of glycatedhemoglobin. The variety of Hb Alc testing methodologies requires a novelcontrol that could be used in various methods and devices for detectingHb Alc levels. In most of the available methods, the first step formeasuring Hb Alc levels is the manual or automatic production of ahemolysate by lysing the red blood cells with a special lytic reagent.Therefore, there is an ongoing need for cellular Hb Alc standards orcontrols that exhibit a similar matrix to that of patient specimens andthat function in the analytical testing phases during an Hb Alc assay.

Currently, there are a number of Hb Alc normal and abnormal controls onthe market. Some of these controls are disclosed in Wu et. al. U.S. Pat.No. 7,247,484 B2; Wu U.S. Pat. No. 6,890,756 B2; Posner et. al. USPatent Publication 2005/0175977; and Ryan et al. U.S. Pat. No. 7,361,513B2; all incorporated herein by reference. The Alc-Cellular controlpreparation described in U.S. Pat. No. 7,361,513 begins by selection ofred blood cells from a suitable subject. The Level 1 (lower or normallevel) is described to be manufactured by utilizing the red blood cellsobtained from a healthy donor with an Alc≦6%. The Level 2 (higher ordiabetic level) was described to be manufactured by utilizing the redblood cells obtained from known diabetic donor with an Alc≧9%. Thesuitable red blood cells were then stabilized and preserved for longterm stability. However, manufacturing diabetic level Alc control usingthe diabetic blood samples, obtained from diabetic donors suffer anumber of serious draw backs. These drawbacks include both inferiorquality and insufficient quantity of diabetic blood samples, as well aseconomical disadvantages. Obtaining natural diabetic blood from diabeticpatients is restricted. Therefore, obtaining sufficient amount ofnatural diabetic blood to meet the growing manufacturing volume of Alccontrol has become an increasing challenge. The availability of diabeticblood sample with a definite range of Alc value is even more difficult.Red Cross eligibility for blood donation states that diabeticindividuals can donate blood only if the individual is under treatmentand the situations are under control. The Alc values of blood samplesobtained from diabetic patients are also inconsistent. According to theAmerican Diabetic Association, any individual with Alc≧6.5 is identifiedas diabetic. The Alc values of blood samples from diabetic patients mayvary from 6.5 to as high as ˜30.0. Further, the lot-to-lot variabilityof the Alc values of the Alc controls manufactured by mixing these bloodsamples are extremely high. In other words, different lots of thediabetic level of Alc control may have significantly different Alcvalues. This inconsistency is much less in the case of normal level dueto abundance of blood sample with Alc≈5-6% range. The Alc values of theblood sample obtained from diabetic patient may appear falsely elevatedor decreased if the blood of the individual donor contains any abnormalhemoglobin variant. A number of clinical studies reported that thepresence of abnormal hemoglobin variants influences Alc values ofhealthy and diabetic patients, see Bry L, Chen P C, Sacks D B. Effectsof hemoglobin variants and chemically modified derivatives on assays forglycohemoglobin. Clin Chem. 2001; 47: 153-163. In general, ion exchangechromatographic and gel electrophoresis methods are affected more thanthe immunoassay or affinity based methods. In case of ion exchangechromatographic method, when the abnormal variant co-elutes with Hb-Alc,then an increase in Alc value is observed. If the abnormal variantco-elutes with A0 (normal hemoglobin), then an apparent decrease in Alcvalue is observed. Therefore, manufacturing Alc control using blood froma donor with unknown hemoglobin composition may cause serious risk inthe accuracy of the Alc values. Rey et al. reported presence of HbSeville[α₂β₂81(EF5)Leu→Phe] causes falsely low Alc value when measuredon ion-exchange chromatography, see Rey T H del, Conde-Sanchez M,Ropero-Gradilla P et al. Hemoglobin Seville [α₂β₂81(EF5)Leu→Phe] asilent phenotypic variant that interferes in hemoglobin Alc measurementby ion-exchange HPLC method. Clin Biochem. 2011; 44: 933-935. Bergman etal. demonstrated that presence of Hb Stockholm [β7(A4)Glu→Asp] causesfalsely low Alc value on Variant II™ chromatography system, see BergmanA C, Beshara S, Byman I, Karim R, Landin B. A new β-chain variant: HbStockholm [(β7(A4)Glu→Asp] causes falsely low HbAlc. Hemoglobin. 2009;33: 137-142. Friess et al. reported that the presence of a novelhemoglobin variant [β66(E10)Lys→Asn] causes a falsely low Alc valuemeasured on cation exchange Tosoh 2.2, see Friess U, Beck A, Kohne E. etal. Novel hemoglobin variant [β66(E10)Lys→Asn], with decreased oxygenaffinity, causes falsely low hemoglobin Alc values by HPLC. Clin Chem.2003; 49: 1412-1415. Chen et. al reported that the Hb-Raleigh[β1Val→Ala] causes false increase in Alc value on ion-exchange, see ChenD, Crimmins D L, Hsu F F, et al. Hemoglobin Raleigh as the cause of afalsely increased hemoglobin Alc in an automated ion-exchange HPLCmethod. Clin Chem. 1998; 44: 1296-1301. Zhu et al. demonstrated that thepresence of HbS in S-β⁺-thalassemia causes a false Alc values on Bio-RadVariant II Turbo, see Zhu Y, Williams L M. Falsely elevated hemoglobinAlc due to S-β⁺-thalassemia interference in Bio-Rad Variant II TurboHbAlc assay. Clin Chim Acta. 2009; 409: 18-20. Frers et al. observedfalsely increased Alc values by HPLC based Tosoh 2.2 for a blood samplethat contained Hb Okayama [β2(NA2)His→Gln], see Frers C R, Dorn S,Schmidt W. et al. Falsely increased HbAlc values by HPLC and falselydecreased values by immunoassay lead to identification of Hb Okayama andhelp in the management of a diabetic patient. Clin Lab. 2000; 46:569-573. Common hemoglobin variants such as Hb S, Hb J, Hb F or Hb E arealso reported by Chu et al. to influence the Alc measurement by Tosoh G7analyzer, Chu C H, Lam H C, Lee J K. et al. Common hemoglobin variantsin southern Taiwan and their effect on the determination of HbAlc byion-exchange high-performance liquid chromatography. J Clin Med Assoc2009; 72: 362-367. Immunoassay method based Alc measurements are alsoknown to be affected by the presence of abnormal hemoglobin variantswhen immune recognition sites of normal Alc or normal hemoglobin aremodified by mutation, see Bry L et al, supra. Blood samples collectedfrom the individuals diagnosed with different diseases are also reportedto cause inaccurate Alc measurements. Consequently, Alc controlmanufactured by using blood samples collected from such patients canintroduce a great deal of inconsistency in Alc values of the controlproduct. Bannon et al.¹¹ and Engbaek et al.¹² reported falsely elevatedAlc values measured by ion-exchange chromatography for the patients withuremia, see Bannon P, Lessard F, Lepage R. Glycated hemoglobin in uremicpatients as measured by affinity and ion-exchange chromatography. ClinChem. 1984; 30: 485-486 and Engbaek F, Christensen S E, Jespersen B.Enzyme immunoassay of hemoglobin Alc: Analytical characteristics andclinical performance for patients with diabetes mellitus, with andwithout uremia. Clin Chem. 1989; 35: 93-97. For such patients,carbamylated derivative of hemoglobin (hemoglobin+urea reaction product)co-elutes with hemoglobin Alc resulting an apparent increase in Alcvalue in ion-exchange chromatographic methods. Suzuki et al. reported anextremely high Alc value (21%) in a patient and reasoned the falseincrease in Alc is due to the acute lymphoblastic leukemia, see SuzukiY, Shichishima T, Yamashiro Y. et al. A patient with acute lymphoblasticleukaemia presenting an extremely high level (21.0%) of HbAlc. AnnalsClin Biochem. 2011; 48: 474-477. Danzig et al. reported that the type 1diabetic patients with glucose-6-phosphatase dehydrogenase deficiencyshowed falsely decreased Alc values, see Danzig J A, Moser J T, BelfieldP. et al. Glucose-6-phosphate dehydrogenase deficiency diagnosed in anadolescent with type 1 diabetes mellitus and hemoglobin Alc discordantwith blood glucose measurement. J. Pediatrics 2011: 849-851. Severalchemical agents used as drug may bind with hemoglobin variants which canaffect Alc measurement. These hemoglobin-drug derivatives can co-elutewith Alc in ion-exchange chromatography causing false result. Likewise,they can interfere with antibody recognition in immunoassay method orchemical recognition in affinity method yielding false results.Evidently, blood samples, obtained from the donors who are undertreatment, are not suitable for manufacturing Alc control with aconsistent Alc value. Aspirin has been identified by a number ofresearchers to cause false elevation of Alc values resulted from HPLCbased analyzers. Aspirin (acetylsalicylic acid) binds with hemoglobinproducing acetylated hemoglobin which co-elutes with Alc in HPLCchromatography resulting falsely elevated Alc, see Nathan D M, Francis TB, Palmer J L. Effect of Aspirin on determination of glycosylatedhemoglobin. Clin Chem. 1983; 29: 466-469; Bridges K R, Schmidt G J,Jensen M. et al. The acetylation of hemoglobin by aspirin. In vitro andin vivo. J Clin Invest. 1975; 56:201-207; Camargo J L, Stifft J, Gross JL. The effect of aspirin and Vitamins C and E on HbAlc assays. Clin ChimActa 2006; 372: 206-209; and Weykamp C W, Penders T J, Siebelder C W M,et al. Interference of carbamylated and acetylated hemoglobin in assaysf glycohemoglobin by HPLC, electrophoresis, affinity chromatography andenzyme immunoassay. Clin Chem. 1993; 39: 138-142. Gross et al.identified ribavirin and peginterferon alfe-2b therapy for hepatitis Cviral infection cause false lowering of Alc values, see Gross B N, CrossB, Foard J C, Falsely low hemoglobin Alc levels in a patient receivingribavirin and peginerferon alfa-2b for hepatitis C. Pharmacotherapy,2009; 29: 121-123. Brown et al. reported false low Alc level for thediabetic patients with chronic kidney disease who were undergoingerythropoietin therapy with epoetin alfa and darbepoetin alfa drugs,Brown J N, Kemp D W, Brice K R. Class effect of erythropoietin therapyon hemoglobin Alc in a patient with diabetes mellitus and chronic kidneydisease not undergoing hemodialysis, Pharmacotherapy, 2009; 29:468-472.Vitamins C and E are also suggested to yield falsely decreased Alcvalues, see Suadek C D, Derr R L, Kalyani R R. Assessing glycemia indiabetes using self-monitoring blood glucose and hemoglobin Alc. ClinRev. 2006; 295:1688-1697 and Schrot R J, Patel K T, Foulis P. Evaluationof inaccuracies in the measurement of glycemia in the laboratory, byglucose meters, and through measurement of hemoglobin Alc. Clin Diabetes2007; 25: 43-49. Blood samples interact inconsistently with variousanalytical methods due to presence of different hemoglobin variants,chemical derivatives of hemoglobin or presence of drugs in the patientblood. A majority of these affect ion-exchange based chromatographicmethods such as HPLC or electrophoresis. However, immunoassay andaffinity based methods are also known to be affected, see Bry L, supra.Inconsistency of the blood samples for Alc control manufacturing canalso be introduced by difference of the ages of the samples obtained.Due to the scarcity, diabetic blood samples might be collected as theybecome available. Therefore, the ages of the blood cells in a collectionof blood packs might be different introducing inferior stability andinconsistent integrity of the cells. As the diabetic donors are rare,the prices of the diabetic blood samples are significantly greater thanthe normal blood samples.

The factors discussed above justify avoiding manufacture high level Alccontrol using natural diabetic blood. The factors also encourage toobtain healthy blood samples and biosynthetically convert it to adiabetic resembling blood sample with high Alc values. Such a conversionto synthetically increase hemoglobin Alc concentration by in vitroglycation of hemoglobin are known in literature, see Posner A H,Reichenbach D L, Hemoglobin isolation and preparation of glycosylatedhemoglobin. US 2005/0175977; Bunn H F, Haney D N, Kamin S. et al. Thebiosynthesis of human hemoglobin Alc. J Clin Invest. 1976; 57: 1652-1659and Spicer K M, Allen R C, Hallett D, Buse M G. Synthesis of hemoglobinAlc and related minor hemoglobins by erythrocytes. In vitro study ofregulation. J. Clin Invest. 1979; 64: 40-48. However, these conversionswere carried out either by pure hemoglobin or by erythrocyte hemolysate.Glycation of hemoglobin within intact red cells are unprecedented. Ryanet al. described two methods of in vitro glycations of hemoglobin withred cells which are less than ideal for manufacturing purpose.¹ Thefirst method proposed by Ryan et al. described a reductive glycation ofred blood

cells with 3% hemoglobin and 0.5% NaCNBH₃. This method yields2-hydroxylized glycated hemoglobin which is not recognized as Alc byion-exchange chromatographic or immunoassay methods. Ryan et al. alsodescribed slow synthesis of Alc by incubating red blood cells with ˜3%glucose at 4-6° C. which takes ˜50 days to achieve desired highconcentration of Alc. It is desirable for efficient manufacturing todevelop processes that can be performed faster.

Many prior art solutions require glycosylation outside of the bloodcells which limit the usefulness of glycosylated materials in controls.Many Alc controls also fail to produce controls that resemble a truepatient blood sample. Such controls cannot be utilized to createuniversal controls, or controls that test multiple parameters of apatient blood sample. The leakage of hemoglobin from the red blood cellsmay render the control ineffective for its intended use, thus requiringthat the red blood cell membranes be preserved to prevent thisundesirable leakage. This can render controls prepared form such redblood cells to not be stable for the desired time frame.

Unlike control products disclosed in the prior art, the presentteachings allow for glycosylation to occur within the red blood cells sothat the synthesis of Alc also occurs within the red blood cells.Unexpectedly, the present teachings include methods whereby the redblood cell membranes are preserved so that such synthesis occurs withoutdamage to the cell membrane. As a further benefit of glycosylationwithin the red blood cells, the resulting control contains a moreuniform population of glycosylated cells. Many Alc controls also fail toproduce controls that resemble a true patient blood sample. Suchcontrols cannot be utilized to create universal controls, or controlsthat test multiple parameters of a patient blood sample. The presentteachings are directed to controls capable of testing multiple elementsof a blood sample, which may include various white blood cellpopulations, nucleated red blood cells, reticulocytes or other bloodcell types. In addition, the present teachings provide for one controlproduct that produces consistent data for a number of hematologyanalyzers, as opposed to requiring a distinct control for each analyzer.As a further benefit of the present teachings, the preservation of thered blood cell membranes results in a shelf-stable product whereby thered blood cells resist hemoglobin leakage for a period of at least about4 months, or longer. The leakage of hemoglobin from the red blood cellsmay render the control ineffective for its intended use, thus requiringthat the red blood cell membranes be preserved to prevent thisundesirable leakage. The present invention addresses one or more of theabove needs by providing improved controls and methods for making thecontrols for testing the Hb Alc level in diabetic blood wherein thecontrols can be prepared in a reasonable time frame with good accuracyand precision and consistency among the standard methods.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the above needs byproviding improved methods for making the controls for testing the HbAlc level in diabetic blood wherein the controls can be prepared tomimic relevant detectable characteristics of diabetic blood in areasonable time frame with good accuracy and precision and attemperatures that do not result in hemolysis. The processing of redblood cells of a starting material can be performed in situ within a redblood cell membrane, and in a manner so that the membrane issubstantially intact from its pre-processing state, thereby avoidinghemoglobin loss and maintaining storage stability (e.g., for a period ofat least one week, two weeks, one month, two months, three months orlonger). Controls according to aspects of the invention are useful inmethods of comparing performance of an instrument against predeterminedknown values for the control. Such methods can be performed for a singleinstrument or across a plurality of different instruments. The presentinvention provides a unique control composition in that it employs as astarting material whole blood from a donor who need not be a diabetic.In fact, starting materials may be free of diabetic whole blood.

The present teachings allow for the for glycosylation of hemoglobin tooccur within the red blood cells so that the synthesis of Alc alsooccurs within the red blood cells; methods whereby the red blood cellmembranes are preserved so that such synthesis occurs without damage tothe cell membrane; controls prepared from such glycosylated hemoglobincontains a more uniform population of glycosylated cells are capable oftesting multiple elements of a blood sample, (which may include variouswhite blood cell populations, nucleated red blood cells, reticulocytesor other blood cell types). In addition, the present teachings providefor one control product that produces consistent data for a number ofhematology analyzers, as opposed to requiring a distinct control foreach analyzer. The preservation of the red blood cell membranes resultsin a shelf-stable product whereby the red blood cells resist hemoglobinleakage for a period of at least about 4 months, or longer.

Unlike control products disclosed in the prior art, the presentteachings allow for glycosylation to occur within the red blood cells sothat the synthesis of Alc also occurs within the red blood cells.Unexpectedly, the present teachings include methods whereby the redblood cell membranes are preserved so that such synthesis occurs withoutdamage to the performance of the cell membrane. As a further benefit ofglycosylation within the red blood cells, the resulting control containsa substantially more uniform population of glycosylated cells. Many Alccontrols also fail to produce controls that resemble a true patientblood sample. Such controls cannot be utilized to create universalcontrols, or controls that test multiple parameters of a patient bloodsample. The present teachings are directed to controls capable oftesting multiple elements of a blood sample, which may include variouswhite blood cell populations, nucleated red blood cells, reticulocytesor other blood cell types. In addition, the present teachings providefor one control product that produces consistent data for a number ofhematology analyzers, as opposed to requiring a distinct control foreach analyzer. As a further benefit of the present teachings, thepreservation of the red blood cell membranes results in a shelf-stableproduct whereby the red blood cells resist hemoglobin leakage and remainstable for an extended period of time that is significantly longer thanthe stability period for untreated human whole blood (e.g., for a periodof at least about 4 months, or longer). The leakage of hemoglobin fromthe red blood cells may render the control ineffective for its intendeduse, thus requiring that the red blood cell membranes be preserved toprevent this undesirable leakage. The newly invented methods focused onthe efficient manufacturing of blood samples with high Alc values fromhealthy blood samples with normal Alc values. These new methods arebased on glycation of hemoglobin within intact red cells. These methodsallow one to circumvent one or more difficulties associated with theprocesses described in the prior art. In vitro glycation can produceblood samples with high Alc values using healthy blood sample withnormal Alc values which are abundant and free of drugs or drugderivatives of hemoglobin. As these normal blood samples are obtainedfrom healthy individuals, they are usually free from interferingabnormal hemoglobin variants. Additionally the in vitro glycation methodalso provides other advantages. The primary advantage is to have controlon the manufacturing process to obtain any amount of high Alc levelglycated blood sample to meet the increasing demand. The standardizedglycation method also allows the manufacturer to eliminate lot to lotvariation of Alc values or blood cell count.

The invention provides a number of advantages over the prior art asdescribed herein. Unlike control products disclosed in the prior art,the present teachings allow for glycosylation to occur within the redblood cells so that the synthesis of Alc also occurs within the redblood cells. Unexpectedly, the present teachings include methods wherebythe red blood cell membranes are preserved so that such synthesis occurswithout damage to the cell membrane. As a further benefit ofglycosylation within the red blood cells, the resulting control containsa more uniform population of glycosylated cells. Many Alc controls alsofail to produce controls that resemble a true patient blood sample. Suchcontrols cannot be utilized to create universal controls, or controlsthat test multiple parameters of a patient blood sample. The presentteachings are directed to controls capable of testing multiple elementsof a blood sample, which may include various white blood cellpopulations, nucleated red blood cells, reticulocytes or other bloodcell types. In addition, the present teachings provide for one controlproduct that produces consistent data for a number of hematologyanalyzers, as opposed to requiring a distinct control for each analyzer.As a further benefit of the present teachings, the preservation of thered blood cell membranes results in a shelf-stable product whereby thered blood cells resist hemoglobin leakage for a period of at least about4 months, or longer. The leakage of hemoglobin from the red blood cellsmay render the control ineffective for its intended use, thus requiringthat the red blood cell membranes be preserved to prevent thisundesirable leakage.

By way of summary, the present invention meets some or all of the aboveneeds by providing in a first aspect a composition comprising red bloodcells in a suspension medium and one or more cis di-ahls; wherein morethan 6 percent by weight of the hemoglobin in the red blood cells isS-Alc glycated. The one or more cis di-ahls comprise one or morecompounds having two active hydrogen containing functional groups onadjacent carbon atoms. Preferably, the suspension medium is an aqueoussuspension medium.

In another aspect, the present invention contemplates a methodcomprising contacting red blood cells in a suspension medium having aconcentration of S-Alc glycated hemoglobin of greater than 6 percent byweight of the hemoglobin in the red blood cells with a sufficient amountof one or more cis di-ahls such that the concentration of S-Alc glycatedhemoglobin in resulting composition as measured by high pressure liquidchromatography, immunoassay and boronate affinity methods is consistent.

In another aspect, the present invention contemplates a cellularhematology control for glycated hemoglobin contained in red bloodcomprising intact red blood cells having a predetermined level ofgreater than about 6 percent by weight of S-Alc glycated hemoglobin andone or more cis di-ahls in a stabilized suspension medium. The cellularhematology control for glycated hemoglobin contained in red blood mayfurther comprise intact red blood cells having a predetermined level ofless than about 6 percent by Weight of S-Alc glycated hemoglobin.

The invention may further comprise a cellular hematology control forglycated hemoglobin contained in red blood cells comprising a) red bloodcells in a stabilized aqueous medium wherein the hemoglobin containsfrom about 4 to about 6 percent by weight of S-Alc glycated hemoglobin;b) red blood cells in a stabilized aqueous medium wherein the hemoglobincontains greater than about 6 and less about X percent by weight ofS-Alc glycated hemoglobin based on the weight of the hemoglobin and oneor more cis di-ahls; and c) red blood cells in a stabilized aqueousmedium wherein the hemoglobin contains greater than about X to about 16percent by weight of S-Alc glycated hemoglobin based on the weight ofthe hemoglobin and one or more cis di-ahls; wherein X is between about 7and 14.

In a preferred embodiment, the cellular hematology control for glycatedhemoglobin contained in red blood cells comprising a) red blood cells ina stabilized aqueous medium wherein the hemoglobin contains from about 4to about 6 percent by weight of S-Alc glycated hemoglobin; b) red bloodcells in a stabilized aqueous medium wherein the hemoglobin containsfrom about 7 to about 9 percent by weight of S-Alc glycated hemoglobinbased on the weight of the hemoglobin and one or more cis di-ahls; andc) red blood cells in a stabilized aqueous medium wherein the hemoglobincontains from about 10 to about 14 percent by weight of S-Alc glycatedhemoglobin based on the weight of the hemoglobin and one or more cisdi-ahls.

In another preferred embodiment the cellular hematology control forglycated hemoglobin contained in red blood cells comprising a) red bloodcells in a stabilized aqueous medium wherein the hemoglobin containsfrom about 4 to about 6 percent by weight of S-Alc glycated hemoglobinbased on the weight of the hemoglobin; and b) red blood cells in astabilized aqueous medium wherein the hemoglobin contains from about 10to about 12 percent by weight of S-Alc glycated hemoglobin based on theweight of the hemoglobin and one or more cis di-ahls.

The invention further contemplates a method for determining the accuracyand reproducibility of the operation of an analytical instrument capableof measuring the glycated hemoglobin levels comprising a) providing acellular hematology control of the invention in a known referencequantity: b) determining the glycated hemoglobin level in said controlof a) with the instrument; and c) comparing the glycated hemoglobinlevels obtained in b) with the known reference quantity.

In another aspect the invention is a composition comprising red bloodcells having a portion of the hemoglobin as acetylated hemoglobin and aportion of hemoglobin as S-Alc glycated hemoglobin, wherein the redblood cells are glycated on the terminal valine amino acid of the Alphaor Beta chain, with the proviso that more than 6 percent by weight ofthe hemoglobin in the red blood cells are acetylated or glycated on theterminal valine amino acid of the Alpha or Beta chain. In yet anotheraspect, the present invention contemplates a method glycating andacetylating red blood cells comprising contacting red blood cells withglucose and acetylsalicylic acid in a suspension medium at about 35° C.to about 40° C. under conditions such that the resulting concentrationof acetylated hemoglobin and S-Alc glycated hemoglobin is greater thanabout 6 percent by weight.

The novel compositions of the invention can be prepared from blooddonors who are healthy, that is blood donors that do not have diabetes.The methods of preparing the controls can be performed in reasonabletime frames that are acceptable in industrial environments and providethe flexibility to provide standards with varying levels of glycatedS-Alc and exhibiting consistency among the standard test methods. Thenewly invented methods focused on the efficient manufacturing of bloodsamples with high Alc values from healthy blood samples with normal Alcvalues. These new methods were based on glycation of hemoglobin withinintact red cells. These methods allow one to circumvent the difficultiesassociated with the processes described by Ryan et al, U.S. Pat. No.7,361,513. In vitro glycation can produce blood samples with high Alcvalues using healthy blood sample with normal Alc values which areabundant and free of drugs or drug derivatives of hemoglobin. As thesenormal blood samples are obtained from healthy individuals, they areusually free from interfering abnormal hemoglobin variants. Additionallythe in vitro glycation method also provides other advantages. Theprimary advantage is to have control on the manufacturing process toobtain any amount of high Alc level glycated blood sample to meet theincreasing demand. The standardized glycation method also allows themanufacturer to eliminate lot to lot variation of Alc values or bloodcell count. The other invented methods takes advantage of catalyticconditions to expedite the glycation of hemoglobin with glucose.Glycation of hemoglobin occurs in two steps. The first step is rapidcondensation of glucose with hemoglobin producing a labile intermediated(L-Alc) followed by a slow rearrangement (Amadori rearrangement)reaction producing stable product, S-Alc. The key of this aspect of theinvention is to accelerate the slow second step by heating or utilizingcatalyst. The novelty of this glycation method is unprecedentedapplication of a series of biocompatible purine based catalysts (e.g.inosine, adenosine) such that the slow in vitro glycation method can beused for manufacturing Alc controls in a reasonable time frame.

DETAILED DESCRIPTION

The invention generally relates to compositions useful in controls fortesting hemoglobin for long term diabetic tendencies. Generally, thecompositions comprise red blood cells having a portion of the hemoglobinas S-Alc glycated hemoglobin, wherein the red blood cells are glycatedon the terminal valine amino acid of the Beta and/or the Alpha chain andone or more cis di-ahls, wherein more than 6 percent by weight of thehemoglobin in the red blood cells are glycated on the terminal valineamino acid of the Beta and/or Alpha chain. S-Alc glycated hemoglobinrefers to stabilized hemoglobin wherein the terminal valine amino acidof the Beta and/or Alpha chain is bonded to the residue of a glucose ormannose molecule. S-Alc glycated hemoglobin is also known as S-Alcglycosylated hemoglobin. The term S-Alc glycated hemoglobin as usedherein includes hemoglobin glycated with glucose and with mannose.Generally, the composition comprises red blood cells having a portion ofthe hemoglobin as acetylated hemoglobin and a portion of hemoglobin asS-Alc glycated hemoglobin, wherein the red blood cells are glycated onthe terminal valine amino acid of the Alpha or Beta chain andacetylated, with the proviso that more than 6 percent by weight of thehemoglobin in the red blood cells are glycated on the terminal valineamino acid of the Alpha or Beta chain or acetylated. The process for thepreparation of the S-Alc glycated hemoglobin using glucose involves a 2step synthetic sequence as described in the equation.

The process for the preparation of the S-Alc glycated hemoglobin usingmannose involves a 2 step synthetic sequence as described in theequation.

The first step is fast and reversible and yields a glycated intermediatewhich is a Shiff base. This first product is often referred to as labileglycated hemoglobin, L-Alc glycated hemoglobin. The second step isirreversible and a very slow step and is known as an Amadorirearrangement. The glycation may occur on the Alc binding site (Val 1 onthe Beta Chain or Alpha Chain) and other non-Alc sites (other Lysresidues). The composition of the invention can contain a range ofconcentration levels of S-Alc glycated hemoglobin in red blood cells.Any concentration level that is useful, for instance in blood controlstandards, may be utilized. Preferably, the concentration of S-Alcglycated hemoglobin in red blood cells is above 6 percent by weight,more preferably about 7 weight percent or greater and most preferablyabout 8 weight percent or greater. Preferably, the concentration ofS-Alc glycated hemoglobin in red blood cells is about 16 weight percentor less, more preferably about 14 percent by weight or less and mostpreferably about 12 percent by weight or less. The normal range ofconcentration of S-Alc glycated hemoglobin in red blood cells found innormal blood donors is about 6 weight percent or below. Above 16 weightpercent is above the range of S-Alc glycated hemoglobin in red blood,cells normally found in diabetic blood. The acetylation of hemoglobin isperformed by reacting acetyl salicylic acid with hemoglobin. Theacetylation occurs on one or more amine sites found in hemoglobin. Thelocation of the acetylation is not entirely clear. Different reports inthe literature have identified different sites for the acetylation. Thisreaction is illustrated by the following formula

The composition of the invention can contain a range of concentrationlevels of acetylated hemoglobin and S-Alc glycated hemoglobin in redblood cells. Any concentration level that is useful, for instance inblood control standards, may be utilized. Preferably, the combinedconcentration of acetylated hemoglobin and S-Alc glycated hemoglobin inred blood cells is above 6 percent by weight, more preferably about 7weight percent or greater and most preferably about 8 weight percent orgreater. Preferably, the combined concentration of acetylated hemoglobinand S-Alc glycated hemoglobin in red blood cells is about 16 weightpercent or less, more preferably about 14 percent by weight or less andmost preferably about 12 percent by weight or less. The normal range ofconcentration of S-Alc glycated hemoglobin in red blood cells found innormal blood donors is about 6 weight percent or below. Above 16 weightpercent is above the range of S-Alc glycated hemoglobin in red bloodcells normally found in diabetic blood. The source of red blood cells ishuman blood. Preferably, the red blood cells are derived from the bloodof a non-diabetic donor and more preferably a healthy donor. Morepreferably, the red blood cells used to synthesize the composition ofthe invention are stabilized red blood cells. Any stabilized red bloodcells useful in manufacturing blood standards may be used to prepare thecompositions of the invention. In a preferred embodiment, the stabilizedred blood cells are stabilized as disclosed in Ryan et al U.S. Pat. No.7,361,513 B2 as disclosed in Column 6 lines 37 to column 11, line 29,incorporated herein by reference. The novel compositions of theinvention can be prepared from blood donors who are healthy, that isblood donors that do not have diabetes. The methods of preparing thecontrols can be performed in reasonable time frames that are acceptablein industrial environments and provide the flexibility to providestandards with varying levels of glycated S-Alc and exhibitingconsistency among the standard test methods. The newly invented methodsfocused on the efficient manufacturing of blood samples with high Alcvalues from healthy blood samples with normal Alc values. These newmethods were based on glycation of hemoglobin within intact red cells.These methods allow one to circumvent the difficulties associated withthe processes described by Ryan et al, U.S. Pat. No. 7,361,513. In vitroglycation can produce blood samples with high Alc values using healthyblood sample with normal Alc values which are abundant and free of drugsor drug derivatives of hemoglobin. As these normal blood samples areobtained from healthy individuals, they are usually free frominterfering abnormal hemoglobin variants. Additionally the in vitroglycation method also provides other advantages. The primary advantageis to have control on the manufacturing process to obtain any amount ofhigh Alc level glycated blood sample to meet the increasing demand. Thestandardized glycation method also allows the manufacturer to eliminatelot to lot variation of Alc values or blood cell count. The otherinvented methods takes advantage of catalytic conditions to expedite theglycation of hemoglobin with glucose. Glycation of hemoglobin occurs intwo steps. The first step is rapid condensation of glucose withhemoglobin producing a labile intermediated (L-Alc) followed by a slowrearrangement (Amadori rearrangement) reaction producing stable product,S-Alc. The key of this aspect of the invention is to accelerate the slowsecond step by heating or utilizing catalyst. The novelty of thisglycation method is unprecedented application of a series ofbiocompatible purine based catalysts (e.g. inosine, adenosine) such thatthe slow in vitro glycation method can be used for manufacturing Alccontrols in a reasonable time frame.

The glycated hemoglobin useful in this invention can be any glycatedhemoglobin prepared utilizing glucose or mannose as glycating sugars.Among preferred processes for glycating hemoglobin are the processesdisclosed herein and in Ryan et at U.S. Pat. No. 7,361,513 B2; andcommonly owned application titled “METHOD OF PREPARING CONTROLS FORGLYCATED HEMOGLOBIN S-Alc DERIVED FROM HEALTHY BLOOD CELLS” filedconcurrently herewith on Nov. 17, 2011 claiming priority from, Ser. No.61/416,623; all incorporated herein by reference.

In one embodiment, glycated hemoglobin is prepared by contacting the redblood cells and glucose in a reaction medium, optionally in the presenceof a compound containing a purine ring having a ribose sugar ligand onthe five membered ring of the purine compound under conditions that aportion of the hemoglobin is converted to S-Alc glycated hemoglobin,wherein the red blood cells are glycated on the terminal valine aminoacid of the Beta chain and/or Alpha chain. In other words, as a resultof this process the concentration of S-Alc glycated hemoglobin isincreased, preferably to a level above 6 percent by weight of thehemoglobin present. In another embodiment the glycated hemoglobin isprepared by contacting red blood cells in a suspension medium containingmannose under conditions such that the concentration of S-Alc glycatedhemoglobin is increased to greater than about 6 percent by weight of thehemoglobin in the red blood cells. Preferably the suspension medium isan aqueous suspension medium. In preferred embodiments, the method isperformed at about ambient temperature, 18° C. to about 23° C.

In general terms, the process for preparing red blood cells containingS-Alc glycated hemoglobin comprises the following steps. In the firststep, a portion of or all of the red blood cells are optionallycontacted with an agent which prevents the hemoglobin in the red bloodcells from converting from the iron II to iron III valence state.Thereafter, the red blood cells are washed with the reaction solutioncontaining glucose or mannose to remove substantially all of thestabilizing solution in which the red blood cells were received. The redblood cells are then contacted with a reaction solution containingmannose, glucose or glucose and a purine ring containing compound havinga ribose sugar based ligand on the five member ring under conditionssuch that a portion of the hemoglobin is converted to S-Alc glycatedhemoglobin, wherein the red blood cells are glycated on the terminalvaline amino acid of the Beta chain and/or Alpha chain. After thereaction mixture has reached the desired stage of glycated hemoglobinconcentration, the red blood cells are washed with the reaction solutionto remove unreacted mannose, glucose or glucose and purine derivative.At this stage the composition generally contains an elevated level ofLabile Alc, the Schiff base described hereinbefore. Thus it is desirableto expose the red blood cells containing and S-Alc glycated hemoglobinand L-Alc hemoglobin to conditions to decompose the L-Alc hemoglobin tohemoglobin and glucose or mannose and to convert a relatively smallportion of the L-Alc hemoglobin to S-Alc glycated hemoglobin. Therelative ratio of L-Alc which decomposes to that which converts to S-Alcglycated hemoglobin is dependent upon the amount of glucose or mannosepresent in the reaction mixture. The formation of the L-Alc Schiff baseis an equilibrium reaction. Preferably this step is performed in theabsence of or at a low concentration of glucose or mannose such thatmost of the L-Alc Schiff base decomposes. Once the desired portion ofL-Alc hemoglobin is decomposed and/or converted to S-Alc glycatedhemoglobin, the red blood cells are placed into a fixing solution for atime sufficient to stabilize the red blood cells. Finally, the red bloodcells are placed in a diluent containing one or more cis di-ahls andstored. Preferably the diluent in which the red blood cells are storedis a suspension medium. Preferably the suspension medium is suitable fordelivering the resulting composition to an analytical instrument foranalysis. The suspension medium contains a cis di-ahl in a sufficientamount such that the amount of S-Alc glycated hemoglobin reported by theboron affinity test is consistent with the amount reported byimmunoassay and high pressure liquid chromatography.

In one aspect a composition of the invention is prepared by contactingthe red blood cells with glucose and acetyl salicylic acid in asuspension medium under conditions that a portion of the hemoglobin isconverted to acetylated hemoglobin and a portion of hemoglobin isconverted to S-Alc glycated hemoglobin, wherein the red blood cells areglycated on the terminal valine amino acid of the Alpha or Beta chain.In general terms, the process for preparing red blood cells containingacetylated hemoglobin and S-Alc glycated hemoglobin comprises thefollowing steps. In the first step, a portion of or all of the red bloodcells are optionally contacted with an agent which prevents thehemoglobin in the red blood cells from converting from the iron II toiron III valence state. Thereafter, the red blood cells are washed withthe reaction solution containing glucose to remove substantially all ofthe stabilizing solution in which the red blood cells were received. Thered blood cells are then contacted with a reaction solution containingglucose and acetyl salicylic acid under conditions such that a portionof the hemoglobin is converted to acetylated hemoglobin and a portion ofhemoglobin is converted to S-Alc glycated hemoglobin, wherein the redblood cells are glycated on the terminal valine amino acid of the Alphaor Beta chain. After the reaction mixture has reached the desired stagethe red blood cells are washed with the reaction solution to removeunreacted glucose and acetyl salicylic acid. At this stage thecomposition generally contains an elevated level of Labile Alc, theSchiff base described hereinbefore. Thus it is desirable to expose thered blood cells containing acetylated hemoglobin and S-Alc glycatedhemoglobin and L-Alc hemoglobin to conditions to decompose the L-Alc tohemoglobin and glucose and to convert a relatively small portion of theL-Alc hemoglobin to S-Alc glycated hemoglobin. The relative ratio ofL-Alc which decomposes to that which converts to S-Alc glycatedhemoglobin is dependent upon the amount of glucose present in thereaction mixture. The formation of the L-Alc Schiff base is anequilibrium reaction. Preferably this step is performed in the absenceof or at a low concentration of glucose such that most of the L-AlcSchiff base decomposes. Once the desired portion of LAlc hemoglobin isdecomposed and converted to S-Alc glycated hemoglobin, the red bloodcells are placed into a fixing solution for a time sufficient tostabilize the red blood cells. Finally, the red blood cells are placedin a diluent and stored. Preferably the diluent in which the red bloodcells are stored is a suspension medium. Preferably the suspensionmedium is suitable for delivering the resulting composition to ananalytical instrument for analysis.

The stabilized red blood cells are typically received in a stabilizedcell wash diluent. Any stabilized diluent utilized for carrying redblood cells may be used as the cell wash diluent. Preferred cell washdiluents used in the process of the invention are described in Ryan etal. U.S. Pat. No. 7,361,513 B2 at Column 7 line 44 to Column 8 line 17as shown in Tables 1 and 2, incorporated herein by reference, andreproduced hereinafter. Certain Hb Alc preparations of the presentinvention utilize the cell wash diluent with the following generalformulation, the solvent or carrier for these compositions is deionizedwater:

Cell Wash Diluent Components Concentration (% w/V) Polyethylene Glycol(FW: 200-50,000) 0-3% EDTA (disodium) 0-3% Magnesium Gluconate(C₁₂H₂₂MgO₁₄•2H₂O) 0-1% Sodium Phosphate dibasic (Na₂HPO₄) 0-2% Glucoseor Mannose 0-8% Methyl Paraben  0-0.2% Inosine  0-0.5% Neomycin SulfateChloramphenicol Sodium Hydroxide (NaOH) Potassium Chloride (KCl)Osmolality (mOsm)The following is an example of the cell wash diluent that has been usedto wash/stabilize red blood cell controls of the present invention.

Cell Wash Diluent Components Concentration (% w/V) Polyethylene Glycol(FW: 20,000) 0.70% EDTA (disodium) 0.70% Magnesium Gluconate(C₁₂H₂₂MgO₁₄•2H₂O) 0.39% Sodium Phosphate dibasic (Na₂HPO₄) 0.27%Glucose or Mannose 0 Methyl Paraben 0.04% Inosine 0.025% NeomycinSulfate 0.04% Chloramphenicol 0.015% Sodium Hydroxide (NaOH) 0.08%Potassium Chloride (KCl) 0.632% pH (Final) 7.0 Osmolality (mOsm) 300

Prior to the glycation step, or acetylation and glycation step, aportion of or all of the red blood cells are optionally contacted withan agent which prevents the hemoglobin in the red blood cells fromconverting from the iron II to iron III valence state. Hemoglobin has Fe(iron) II at the center which is utilized to bind and carry oxygen inthe blood stream. When the Fe at the center of the hemoglobin is in theII ionic state the red blood cells retain a red color. The Fe II canundergo oxidation to form Fe III which causes the red blood cells toturn brown. Thus it is desirable to prevent the oxidation of the Fe IIsites to Fe III. This is achieved by contacting the red blood cells withan agent that prevents oxidation of the Fe in the center of thehemoglobin. Any such agent which prevents this oxidation may beutilized. It is believed that the antioxidation agent bonds to the Fe atthe center of the hemoglobin which prevents the Fe from oxidizing.Examples of such agents include carbon monoxide (CO) and the like. Apreferred anti oxidation agent is carbon monoxide. The antioxidationagent is preferably contacted with the red blood cells while the redblood cells are located in a cell wash diluent, preferably in astabilized a cell wash diluent. Where the antioxidation agent is a gas,such as carbon monoxide, the gaseous antioxidation agent is bubbledthrough the cell wash diluent containing the red blood cells. Where theantioxidation agent is a liquid or solid it is mixed into the cell washdiluents. The antioxidation agent is contacted with the red blood cellsunder conditions such that a sufficient number of the Fe II cites at thecenter of the hemoglobin in the red blood cells are bonded to orassociated with antioxidants such that the red blood cells retain a redcolor. Preferably about 40 mole percent or greater of the Fe II sitesare bonded to or associated with an antioxidant, more preferably about45 mole percent or greater and most preferably about 49 mole percent orgreater. Preferably about 60 mole percent or less of the Fe II sites arebonded to or associated with an antioxidant, more preferably about 55mole percent or less and most preferably about 51 mole percent or less.If too many of the Fe II sites in the hemoglobin of the red blood cellsare bonded to or associated with antioxidant molecules, untreated redblood cells in cell wash diluents can be added to the treated red bloodcells to provide the desired concentration of antioxidant modifiedhemoglobin. In the preferred embodiment wherein the antioxidant iscarbon monoxide, the carbon monoxide is bubbled through the cell washdiluent containing the red blood cells until the amount of the Fe IIsites of the hemoglobin are bonded to or associated with antioxidantmolecules of the red blood cells is greater than desired. Thereafter, aportion of untreated red blood cells in cell wash diluent are added tothe treated red blood cells to provide the desired concentration ofantioxidant treated hemoglobin in the red blood cells.

The red blood cells are separated from the stabilized cell wash diluent.The separation is performed using any known method of separating solidmaterials from liquid media, provided the cells are not damaged.Examples of preferred separation techniques include filtration,centrifuging the medium with blood cells contained therein, dialysis andthe like. Preferably, the contacting is performed as a series of washeswherein the red blood cells are separated from the wash solution bymeans known in the art, for example filtration or centrifugation.Thereafter, the red blood cells are then contacted with the medium usedfor the glycation reaction.

A cell wash diluent may be utilized as the reaction medium, included inthe reaction media useful are those disclosed in Table 4 of Ryan et al.U.S. Pat. No. 7,361,513 B2 at Column 9, lines 24 to 45 which isreproduced as Table 3 hereinafter. Preferably, the reaction medium isaqueous based and acts as a suspension medium for the red blood cells.In this embodiment, the reaction medium can also be referred to as asuspension medium. The reaction medium is based on deionized water.

TABLE 3 Reaction Medium Components Concentration (% w/V) PolyethyleneGlycol (FW: 200-50,000) 0-3%  EDTA (disodium) 0-3%  Magnesium Gluconate(C₁₂H₂₂MgO₁₄•2H₂O) 0-1%  Sodium Phosphate dibasic (Na₂HPO₄) 0-2% Glucose or Mannose 0-8%  Methyl Paraben 0-0.2% Inosine 0-0.6% NeomycinSulfate 0-0.2% Chloraphenicol 0-0.2% Potassium Chloride (KCl) 0-1.5 Sodium Fluoride (NaF) 0-0.5% Ciprofloxacin* 0-0.1% Sodium Hydroxide(NaOH) 0-0.5% pH (final) 6.0-8.0     Osmolality (mOsm) 250-350    *Final

Addition.

The composition in Table 3 may also used in the stabilizing media of thefinal composition of the invention, and the ingredients marked finaladdition are added after completion of the glycation. The reactionmedium further contains bovine serum albumin, which is present for thepurpose of anticoagulation and anti-hemolysis. A sufficient amount ofbovine serum albumin is present in the reaction medium to prevent thecoagulation of the cells and hemolysis of the cells. The lower limit onthe concentration of bovine serum albumin in the reaction medium islowest concentration at which hemolysis or coagulation are prevented.Preferably, bovine serum albumin is present in the reaction medium in aconcentration of about 1 percent by weight or greater and morepreferably about 3 percent by weight or greater. The upper limit on theconcentration of bovine serum albumin in the reaction medium is thatconcentration at which no further reduction of coagulation and hemolysisis possible. Preferably, bovine serum albumin is present in the reactionmedium in a concentration of about 10 percent by weight or less, morepreferably about 5 percent by weight or less and most preferably about 4percent by weight or less. The reaction medium further contains glucoseor mannose in sufficient amount to glycate, or glycated and acetylatethe hemoglobin to the desired level. The lower limit on theconcentration of glucose or mannose in the reaction medium is thatconcentration at which hemoglobin is glycated at a reasonable rate.Preferably, glucose is present in the reaction medium in a concentrationof about 3 percent by weight or greater and more preferably about 5percent by weight or greater. The upper limit on the concentration ofglucose in the reaction medium is about 10 percent. Preferably, glucoseis present in the reaction medium in a concentration of about 8 percentby weight or less, more preferably about 7 percent by weight or less andmost preferably about 6 percent by weight of less. When Mannose is theglycating agent, the reaction medium further contains mannose insufficient amount to glycate the hemoglobin to the desired level. Thelower limit on the concentration of mannose in the reaction medium isthat concentration at which hemoglobin is glycated at a reasonable rate.Preferably, mannose is present in the reaction medium in a concentrationof about 4 percent by weight or greater and more preferably about 5percent by weight or greater. The upper limit on the concentration ofmannose in the reaction medium is about 8 percent and most preferablyabout 6 percent by weight of less. The reaction medium may furthercontains acetyl salicylic acid in sufficient amount to acetylate thehemoglobin to the desired level. The lower limit on the concentration ofacetyl salicylic acid in the reaction medium is that concentration atwhich hemoglobin is acetylated at a reasonable rate. Preferably, acetylsalicylic acid is present in the reaction medium in a concentration ofabout 0.1 percent by weight or greater, more preferably about 0.2percent by weight or greater and most preferably about 0.24 percent byweight or greater. The upper limit on the concentration of acetylsalicylic acid in the reaction medium is the solubility of the acetylsalicylic acid in the reaction medium. Preferably, acetyl salicylic acidis present in the reaction medium in a concentration of about 0.4percent by weight or less, more preferably about 0.3 percent by weightor less and most preferably about 0.26 percent by weight of less. Theconcentration of red blood cells in the reaction medium is chosen suchthat the hemoglobin contained therein can be glycated and acetylated ata reasonable rate. If the concentration is too high the reaction rate istoo slow. The concentration of red blood cells in the reaction medium ischosen such that the hemoglobin contained therein can be glycated at areasonable rate. If the concentration is too high the reaction rate istoo slow.

In one embodiment, the red blood cells are contacted with glucose in thepresence of a purine compound having a ribose ligand bonded to the fivemembered ring of the purine compound. Any purine compound having aribose sugar ligand bonded to the five membered ring of the purinecompound that catalyzes the reaction of glucose with hemoglobin to formSAlc glycated hemoglobin may be utilized in this step. In a preferredembodiment the ribose ligand is bonded to a nitrogen atom on the fivemembered ring of the purine compound. The purine compound may containone or more substituents on one or more of the purine rings. Anysubstituents that do not interfere with catalyzing the reaction ofglucose with hemoglobin may be present on the purine ring. Preferablysuch substituents are capable of hydrogen bonding with groups on thehemoglobin molecule. Preferably such substituents are capable ofhydrogen group abstraction. The substituents are preferably are selectedfrom the group of amines, hydroxyls, thiols, amides, carboxylic acids,carboxylic amides, and oxygen doubly bonded to a carbon on one of therings. Preferably such substituents are selected from the group ofprimary amines and oxygen doubly bonded to a carbon on one of the rings.Preferably the substituents are located on the six membered ring. Theribose sugar ligand can have one or mores substituents bonded thereto.Preferably two or more of the carbons on the ribose sugar ring havehydroxyl groups or oxygen atoms bonded thereto and most preferablyhydroxyl groups. The ribose sugar ligand may have bonded to one or moreoxygen atoms a phosphate group. In a preferred embodiment the purinecompound corresponds to the formula

wherein R¹, R², R³ and R⁴ are separately in each occurrence hydrogen ora substituent that does not interfere with the reaction of glucose withhemoglobin. Preferably, R¹ and R² are separately in each occurrencehydrogen or functional groups which are capable of hydrogen bonding toparts of hemoglobin. More preferably, R¹ and R² are separately in eachoccurrence hydrogen or selected from the group of amines, hydroxyls,thiols, amides, carboxylic acids, carboxylic amides, and oxygen doublybonded to a carbon on the six membered ring. Even more preferably, R¹and R² are separately in each occurrence hydrogen or selected from thegroup of primary amines and oxygen doubly bonded to a carbon on the sixmembered ring. Preferably, R³ is separately in each occurrence hydrogenor a phosphate group (the phosphorous of the phosphate group is bondedto oxygen) and most preferably hydrogen. Included among preferred purinecontaining compounds are inosine, adenosine, guanosine, xanthosine,2-aminoadenosine, AMP (Adenosine monophosphate) and Cyclic AMP. Evenmore preferred purine containing compounds are inosine and adenosine.The preferred compounds correspond to the following formulas:

The purine compounds are utilized in the step of contacting red bloodcells with glucose in a sufficient amount to catalyze the glycation ofhemoglobin. Preferably the purine compounds are present in an amount ofabout 0.1 percent by weight or greater based on the weight of thereaction medium, such as an aqueous suspension medium, and morepreferably about 0.2 percent by weight or greater. Preferably, thepurine compounds are present in an amount of about 0.5 percent by weightor less based on the weight of the reaction medium, such as an aqueoussuspension medium, and more preferably about 0.4 percent by weight orless.

In a preferred embodiment, the concentration of red blood cells is thesame concentration as found in human blood. It is preferred to monitorthe concentration of hemoglobin in the reaction medium. Preferably theconcentration of hemoglobin in the reaction medium is about 9 grams perdeciliter (g/dL) or greater and more preferably about 10 grams perdeciliter (g/dL) or greater. Preferably the concentration of hemoglobinin the reaction medium is about 12 grams per deciliter (g/dL) or lessand more preferably about 11 grams per deciliter (g/dL) or less. Theconcentration can also be expressed as the concentration of red bloodcells which is preferably about 4.0 million red blood cells permicroliter of reaction medium or greater and more preferably about 5.0millions red blood cells or greater. Preferably the concentration of redblood cells in the reaction medium is 7.0 million of red blood cells permicroliter of reaction medium or less.

In the embodiment wherein glucose is the glycating agent, and whereacetyl salicylic acid is optionally present, the reaction medium isexposed to elevated temperatures for sufficient time to achieve thedesired concentration of S-Alc glycated hemoglobin. The temperature ischosen such that the glycation reaction proceeds at a reasonable rateand not so high that the red blood cells are damaged. Preferably, thereaction is performed at a temperature of about 35° C. or greater andmore preferably at a temperature of about 37° C. or greater. Preferably,the reaction is performed at a temperature of about 40° C. or less andmore preferably at a temperature of about 38° C. or less. Where mannoseis the glycating agent these temperatures may be utilized although it ispreferred to glycate with mannose at lower temperatures down to roomtemperature.

In the embodiment wherein mannose is the glycating agent, the reactionmedium is exposed to temperatures for at which the desired concentrationof S-Alc glycated hemoglobin can be reached in a reasonable time period.The temperature is chosen such that the glycation reaction proceeds at areasonable rate and not so high that the red blood cells are damaged.Preferably, the reaction is performed at a temperature of about 18° C.or greater and more preferably at a temperature of about 23° C. orgreater. Preferably, the reaction is performed at a temperature of about40° C. or less, and more preferably at a temperature of about 38° C. orless, even more preferably at a temperature of about 25° C. or less andmore preferably at a temperature of about 23° C. or less.

Generally, the glycation reaction is performed for a time sufficient toachieve the desired concentration of S-Alc glycated hemoglobin. In apreferred embodiment, the process of the reaction is monitored by ananalytical technique which measures the concentration of the S-Alcglycated hemoglobin in the reaction medium. Any analytical techniquewhich measures the concentration of S-Alc glycated hemoglobin can beused. Among preferred analytical techniques is high pressure liquidchromatography (HPLC). In a preferred embodiment, the high pressureliquid chromatography is performed utilizing an Alc 2.2 Plus or G8Analyzer available from TOSOH Bioscience. Preferably the reaction isallowed to proceed until the concentration of S-Alc glycated hemoglobin,or S-Alc glycated hemoglobin and acetylated hemoglobin, in the red bloodcells is greater than about 6 percent by weight based on theconcentration of hemoglobin in the red blood cells and more preferablyabout 7 or greater. Preferably, the reaction is allowed to proceed untilthe concentration of S-Alc glycated hemoglobin, or S-Alc glycatedhemoglobin and acetylated hemoglobin, in the red blood cells is about 16percent by weight or less based on the concentration of hemoglobin inthe red blood cells and more preferably about 14 percent by weight orless.

Where glucose alone is the glycating agent the reaction time is definedin Ryan et al. U.S. Pat. No. 7,361,513 B2. Wherein glucose in thepresence of a purine compound, or acetyl salicylic acid, is theglycating agent or Mannose is the glycating agent and elevatedtemperatures are used, the reaction is performed for a time period suchthat the desired concentration of S-Alc glycated hemoglobin in the redblood cells is reached. Preferably, the reaction time is 20 hours orgreater and more preferably 22 hours or greater. Preferably the reactiontime is 30 hours or less and more preferably 24 hours of less. Thereaction time is dependent on the reaction temperature and the mannoseor glucose and purine compound or acetyl salicylic acid concentrations.At lower temperatures the reaction times are longer and at highertemperatures the reaction time is shorter. For this reason it isimportant to monitor the concentration of S-Alc glycated hemoglobin inthe reaction mixture.

Where mannose is the glycating agent and the glycation occurs at aboutroom temperature, the reaction is performed for a time period such thatthe desired concentration of S-Alc glycated hemoglobin in the red bloodcells is reached. Preferably, the reaction time is 4 days or greater andmore preferably 6 days or greater. Preferably the reaction time is 8days or less. The reaction time is dependent on the reaction temperatureand the mannose concentration. At lower temperatures the reaction timesare longer and at higher temperatures the reaction time is shorter. Forthis reason it is important to monitor the concentration of S-Alcglycated hemoglobin in the reaction mixture.

The reaction can be performed in the presence of air and can beperformed in either an open or closed vessel. A closed vessel ispreferred as it provides for greater control of the reactionenvironment.

Once the desired concentration is achieved the reaction medium isallowed to cool, preferably to room temperature, about 18° C. to about25° C., and preferably about 22° C. Thereafter, the glucose, optionalacetyl salicylic acid, or mannose is removed from the reaction mixtureto prevent further glycation. Any method of removing the glucose,optional acetyl salicylic acid, or mannose known to the skilled artisanmay be utilized. Preferably, the red blood cells are removed from thereaction medium by a known separation technique, such as filtration,centrifuging or dialysis. The red blood cells are preferably washed witha diluent. Preferably the diluent utilized is a diluent as described inTable 3. In order to insure that all of the glucose or mannose isremoved from the red blood cells, it is preferred to wash the red bloodcells multiple times with the diluents. The red blood cells are washedwith the diluent a sufficient number of times to remove substantiallyall of the glucose or mannose from the red blood cells. Generally, threewashes results in a sufficient removal of the glucose or mannose. In apreferred embodiment the diluent contains bovine serum albumin,preferably in a concentration of about 3 percent to about 5 percent.

The red blood cells recovered from the glycation process generallycontain a higher concentration of minor by-products, such as L-Alchemoglobin, than typically found in diabetic blood. Thus it ispreferable to adjust the level of the minor by-products in the blood bya deglycation step. In this step the red blood cells are dispersed in adiluent. Any diluent known to one skilled in the art may be used.Generally any diluent utilized for the first step may be used with theexception that mannose, glucose or glucose and purine compounds are notpresent. During the deglycation process the red blood cells dispersed ina diluent are exposed to an elevated temperature for a period of timesuch that the concentration of minor byproducts is adjusted to be in thenormal range. Normal range, as used in this context, means theconcentration of the minor byproducts are within the range minorbyproducts that are found in naturally occurring red blood cells havingthe same concentration of S-Alc glycated hemoglobin. Any temperaturewhich allows the conversion of the minor byproducts to an acceptablerange in a reasonable time frame and which does not harm the red bloodcells may be utilized. If the temperature is too low the reaction timeis too long. If the temperature is too high the red blood cells can bedamaged, such as through hemolysis. In the embodiment wherein glucose isthe glycating agent, the deglycation is preferably performed at ambient(room temperature) or above and more preferably at a temperature ofabout 25° C. or greater and more preferably at a temperature of about30° C. or greater. Preferably, the deglycation is performed at atemperature of about 40° C. or less and more preferably at a temperatureof about 38° C. or less. Where the glycation agent is mannose, thedeglycation is performed preferably at ambient (room temperature) 18° C.or above. Preferably, the deglycation is performed at a temperature ofabout 40° C. or less, and more preferably at a temperature of about 38°C. or less, even more preferably about 30° C. or less, even morepreferably about 25° C. or less and most preferably about 23° C. orless. The deglycation time period is chosen to achieve the desiredconcentration of minor by-products. In a preferred embodiment, theconcentration of L-Alc glycated hemoglobin is used to determine if theconcentration of minor by-products is in an acceptable range. The L-Alcglycated hemoglobin is generally dissociated in this step. In apreferred embodiment, the deglycation process is monitored by ananalytical technique which measures the concentration of the L-Alcglycated hemoglobin in the reaction medium. Any analytical techniquewhich measures the concentration of L-Alc glycated hemoglobin can beused. Among preferred analytical techniques useful is high pressureliquid chromatography as described herein before. Preferably, thedeglycation is performed under conditions such that the resultingconcentration of L-Alc glycated hemoglobin is about 4 percent by weightor greater and most preferably about 4.2 percent by weight or greater.Preferably, the deglycation is performed under conditions such that theresulting concentration of L-Alc glycated hemoglobin is about 5 percentby weight or less and most preferably about 4.8 percent by weight orless.

The deglycation time is chosen such that the desired concentration ofL-Alc is achieved. Preferably, the deglycation time is about 20 hours orgreater and more preferably about 22 hours or greater. Preferably thedeglycation time is about 30 hours or less and more preferably about 24hours of less. The deglycation time is dependent on the reactiontemperature. At lower temperatures the reaction times are longer and athigher temperatures the reaction time is shorter. For this reason it isimportant to monitor the concentration of L-Alc hemoglobin in thereaction mixture. The deglycation can be performed in the presence ofair and can be performed in either an open or closed vessel. A closedvessel is preferred as it provides for greater control of thedeglycation environment.

Once the desired concentration of L-Alc glycated hemoglobin is achievedthe reaction medium is allowed to cool, if necessary, preferably to roomtemperature, about 22° C. Preferably, the red blood cells are removedfrom the reaction medium by a known separation technique, such asfiltration and/or centrifugation. The red blood cells are preferablywashed with a diluent. Preferably the diluent utilized is a diluent asdescribed in Table 3. It is preferred to wash the red blood cellsmultiple times with the diluent. The red blood cells are washed with thediluent a sufficient number of times to remove substantially all of theunwanted by-products. Generally three washes result in a sufficientremoval of the unwanted by-products. Preferably the stabilizing diluentis water based, aqueous, and the stabilizing diluent suspends the redblood cells, that is the final composition is located in an aqueoussuspension medium.

Thereafter, the red blood cells are contacted with a fixing solution asdisclosed in Ryan et. al. U.S. Pat. No. 7,361,513 at Column 8 lines 19to 44, incorporated herein by reference. The red blood cells are fixedusing a known cell fixing compound or composition. The cell fixingcompound or composition serves to strengthen the cell membrane and tominimize the change in mean cell volume (MCV), thus to prevent thehemolysis of red blood cells. In addition, the fixation allows the cellfixing compound or composition to cross link hemoglobin, which createsmore homogeneity and stability of chemical charge for hemoglobin andenhances its HPLC performance during long-term stability testing. Thefixative may include, but is not limited to, one or more of an aldehyde,oxazolidine, alcohol, cyclic urea, or the like. Examples of suchfixatives include, without limitation, formaldehyde, glutaraldehyde,diazolidinyl urea (DU), imidazolidinyl urea (IDU), dimethylol urea,dimethylol-5,5-dimethylhydantoin, 2-bromo-2-nitropropane-1,3-diol;quaternary adamantine, hydroxyl-methyl-1-aza-3,7-dioxabicyclo(3.3.0)octane, sodium hydroxymethyl glycinate, and mixtures thereof, orthe like. Other fixatives may be used such as those disclosed in U.S.Pat. Nos. 5,196,182; 5,262,327; 5,460,797; 5,811,099; 5,849,517;6,221,668; 5,529,933; 6,187,590 (incorporated herein by reference). Theappropriate fixative reagent is selected based upon the cell attributeto be evaluated by a hematology analyzer. In a preferred embodiment, thepresent invention utilizes a fixation process to generate the controlcomposition. In one particular embodiment of the present invention, theblood cell fixation begins by contacting the washed blood cells with afixative reagent. A more preferred cell fixation compound isglutaraldehyde. A cell fixation procedure using glutaraldehyde isperformed between cell filtration and cell final wash. A general cellfixation procedure includes the following steps: adjusting the count offiltered RBC to approximately 4±0.2 M/μL, using the cell wash diluentdescribed in Table 2; measure the total volume of RBC; measuring thesame volume of cell wash diluent in another container. Add 0.1-4.0 mL/Lof glutaraldehyde (25 percent stock) to the diluent and mixing well;mixing the RBC solution and glutaraldehyde solution thoroughly; andplace the mixed solution at room temperature for 24 hours before finalcell washing. An example of the cell fixation procedure included thepreparations of a 4.0 M/μL RBC solution and a 0.8 mL/L glutaraldehydesolution and a quick mixing of the two solutions at room temperature.

After the cell fixation is complete, the red blood cells are separatedfrom the fixation compound or composition using known separationtechniques, such as filtration, centrifuging, dialysis or a combinationthereof. Thereafter, red blood cells are washed using a cell washdiluent such as a composition as described in table 3. Multiple washesare generally utilized. After every wash the red blood cells areseparated from the cell wash diluent.

After washing, the red blood cells are suspended in a final stabilizingmedium (diluent). The final stabilizing diluent is used to stabilize thevarious controls of the present invention. The final stabilizing diluentis preferably an aqueous based medium. It is desirable for the finalstabilizing diluent to possess the following attributes: (1) tostabilize the value of, percentage of S-Alc glycated hemoglobin at bothclosed-vial and open-vial modes; and (2) to prevent red blood cellhemolysis. Similar to the cell wash diluent, the final stabilizingdiluent includes appropriate cell stabilizers (e.g. magnesium gluconate,EDTA and PEG), cell metabolites (e.g. inosine and glucose), buffers(e.g. sodium phosphate dibasic and/or monobasic),antibiotics/antimicrobial agents (e.g. neomycin sulfate andchloramphenicol), and anti-fungal agents (e.g. methyl paraben). Inaddition, final stabilizing diluent contains one or more of thefollowing components: glucose, and sodium. The formulations of finalstabilizing diluents vary slightly depending upon the desired level ofS-Alc hemoglobin in the different levels of S-Alc hemoglobin controls(such as Level I or II). The final stabilizing diluent does not have tocontain all of the components listed in Table 3, but will include atleast as many of the components listed in Table 3 to provide thedesired, S-Alc hemoglobin levels. In one preferred embodiment thesuspension medium contains from about 0.3 to about 0.6 percent by weightof glucose based on the weight of the suspension medium.

The stabilized suspension of red blood cells may contain one or more cisdi-ahls. A cis di-ahl is a compound having two ahl groups on adjacentcarbon atoms. An ahl group is a functional group containing an activehydrogen atom. Thus a cis di-ahl is a compound having functional groupscontaining active hydrogen atoms on adjacent carbon atoms. Functionalgroups having active hydrogen atoms are well known to those skilled inthe art. Preferred functional groups containing active hydrogen atomsinclude hydroxyl, amino, thiol and carboxylate functional groups; withamino, thiol and hydroxyl being more preferred and hydroxyl mostpreferred. Any cis di-ahl can be used herein that binds to the resinused in the boronate affinity analytical method and which does not alterthe results of the other analytical test used for measuring the S-Alclevels in red blood cell hemoglobin. Preferably the one or more cisdi-ahls are cell impermeable so that they do not swell or increase thecell volume of the red blood cells. Preferably the one or more cisdi-ahls do not react with mannose, glucose or functional groups onhemoglobin. Preferred classes of di-ahls include cis diols, cisdiamines, cis dicarboxylic acids, cis thiols, compounds with cis aminoand hydroxyls, compounds with cis thiol and hydroxyl groups, andcompounds with cis amino and thiol groups. More preferred classes of cisdi-ahls are cis-diols. Preferred cis di-ahls include sugars (fructose),reduced sugars (sorbitol, manitol, xylitol), serine, cysteine anddithiothreitol. More preferred cis di-ahls include sorbitol, fructoseand manitol with sorbitol most preferred. The cis di-ahls are added tothe stabilized suspension medium (diluent) as described hereinbefore.The amount of cis di-ahl used is selected such that the amount of S-Alcglycated hemoglobin reported by the boron affinity test is consistentwith the amount reported by immunoassay and high pressure liquidchromatography. Consistent as used in this context means the reportedpercentage of S-Alc glycated hemoglobin reported by the boron affinitytest is no more than 2 percentage points different than reported byimmunoassay and high pressure liquid chromatography, and more preferablyno more than 1 percentage point. The amount of cis di-ahl in thestabilized diluent, preferably an aqueous suspension medium, is about1.0 percent by weight or greater based on the weight of the stabilizeddiluent, more preferably about 2.0 percent by weight or greater and mostpreferably about 3.0 percent by weight or greater. The amount of cisdi-ahl in the stabilized diluent, preferably an aqueous suspensionmedium, is about 10.0 percent by weight or less based on the weight ofthe stabilized diluent, more preferably about 8.0 percent by weight orless and most preferably about 6.0 percent by weight or less.

As discussed herein there are several washing steps. Typically, washingwill employ contacting the cells with a suitable solution, preferably abuffered solution, and most preferably a suitable isotonic washsolution. The wash solution, which generally will be substantiallyisotonic, may include any of a number of ingredients in a deionizedand/or distilled water base. For example, the wash solution may includeat least one or more, more preferably two or more, still more preferablythree or more, still more preferably four or more and still even morepreferably all of the following ingredients: a fungicide; anantimicrobial; a surfactant; a buffer; a metal chelating agent; a cellnutrient; or an agent for maintaining tonicity. For example, in oneparticular embodiment of the present invention, the relative amounts ofthe above ingredients may be as follows: a fungicide of up to about 5parts; an antimicrobial of up to about 5 parts; a surfactant rangingfrom about 5 parts to about 20 parts; a buffer ranging from about 5parts to about 30 parts, a metal chelating agent ranging from about 25parts to about 50 parts; a cell nutrient of up to about 5 parts; and anagent for maintaining tonicity in about 15 parts to about 35 parts. Thewash solution may also contain other ingredients as described in U.S.Pat. No. 5,858,790 or U.S. Pat. No. 6,187,590 (incorporated by referenceherein). The isotonic wash solution may also include ingredients thatact as a homolysis inhibitor, an aggregating agent, a cell stabilizer,an antioxidant, or a mixture thereof. By way of example, one possiblewash solution may include about 40 mg percent Methyl Paraben, about 300mg percent polyethylene glycol (PEG)—(molecular weight about 20,000);about 1675 mg percent ethylenediaminetetraacetic acid (EDTA); about 933mg percent magnesium gluconate; about 639 mg percent sodium phosphatedibasic anhydrous, about 25 mg percent adenosine, about 25 mg percentInosine; about 40 mg percent neomycin sulfate; and about 15 mg percentchloramphenicol.

The fixation may take place at any temperature and preferably at roomtemperature, about 18° C. to 25° C. in a preferred embodiment of thepresent invention, the fixed red blood cells are washed out of thefixative reagent and re-suspended into a suitable suspension medium. Thecells can be washed out of the fixative reagent with a buffered isotonicsolution. In one preferred embodiment, after contact with the hypotonicsolution, the cells are re-suspended in a diluent. In one preferredembodiment, the cells are resuspended, for instance, in a phosphatebuffered solution containing polyethylene glycol 20,000 (PEG),ethylenediamine tetraacetic acid (EDTA) and magnesium gluconate with 2percent bovine serum albumin.

The red blood cells processed as described herein before can be utilizedto prepare controls used to calibrate instruments used to measure thelevel of certain components in blood. The red blood cells prepared asdescribed herein are preferably used to calibrate instruments thatmeasure the level of S-Alc glycated hemoglobin or S-Alc glycatedhemoglobin and acetylated hemoglobin. Preferably controls containing thered blood cells of the invention are used to calibrate apparatus whichanalyze red blood cells by high pressure liquid chromatography,immunoassay and boronate affinity. In a preferred embodiment, theconcentration of S-Alc glycated hemoglobin, or S-Alc glycated hemoglobinand acetylated hemoglobin, in the red blood cell controls of theinvention as measured by high pressure liquid chromatography,immunoassay and boronate affinity methods are consistent, morepreferably the measured concentration of S-Alc glycated hemoglobin, orS-Alc glycated hemoglobin and acetylated hemoglobin, in the red bloodcell controls of the invention as measured by high pressure liquidchromatography, immunoassay and boronate affinity methods are within arange of about 2 percent by weight or less of each other and morepreferably about 1 percent by weight or less.

The controls of the invention comprise the red blood cells of theinvention in an appropriate suspension medium. The controls are used forcellular hematology controls. The suspension medium can be anysuspension medium utilized for red blood cell controls. The suspensionmedium can comprise the compositions described hereinbefore as the finalstabilizing medium. The suspension media are preferably water based. Thecontrols contain one or more sets of red blood cells in a suspensionmedium having a known concentration of S-Alc glycated hemoglobin and oneor more cis di-ahls. Preferably, the controls contain two or more setsof red blood cells in a suspension medium having a known concentrationof S-Alc glycated hemoglobin, or S-Alc glycated hemoglobin andacetylated hemoglobin, and one or more cis di-ahls. In a more preferredembodiment, the controls comprise two or three sets of red blood cellsin a suspension medium having a known concentration of S-Alc glycatedhemoglobin and one or more cis di-ahls. A combination of two or moresets of red blood cells can be assembled into kits. Preferably, thecontrols contain one set of red blood cells with S-Alc glycatedhemoglobin in the normal range, non-diabetic range, of about 4 to 6percent by weight. Preferably, the controls contain one or more sets ofred blood cells having concentrations of S-Alc glycated hemoglobin, orS-Alc glycated hemoglobin and acetylated hemoglobin, of greater thanabout 6 percent by weight, more preferably about 7 percent by weight orgreater and most preferably about 8 percent by weight or greater.Preferably, the controls contain one or more sets of red blood cellshaving concentrations of S-Alc glycated hemoglobin, or S-Alc glycatedhemoglobin and acetylated hemoglobin, of about 16 percent by weight orless, more preferably about 14 percent by weight or less and mostpreferably about 12 percent by weight or less. The sets of the controlshaving greater than about 6 percent by weight of glycated hemoglobincontain one or more cis di-ahls. The controls which contain S-Alcglycated hemoglobin in the normal range, 6 percent or less, do notrequire the presence of a cis di-ahl.

The controls of the invention contain one or more separate suspensionsof red blood cells, and preferably two of more. The concentration ofS-Alc glycated hemoglobin, or S-Alc glycated hemoglobin and acetylatedhemoglobin, in each suspension, wherein the concentration is above thenormal concentration, is within the ranges set out hereinbefore. Wherethere are two or more sets of red blood cells in the control, theconcentrations of S-Alc glycated hemoglobin, or S-Alc glycatedhemoglobin and acetylated hemoglobin, are different in each set. Whenthere are two suspensions having S-Alc glycated hemoglobin, or S-Alcglycated hemoglobin and acetylated hemoglobin, concentrations above thenormal range, the concentrations of S-Alc glycated hemoglobin, or S-Alcglycated hemoglobin and acetylated hemoglobin, in the two suspensionsare related such that the first has a concentration of greater than 6 toabout X and the second has a concentration of greater than X to about16. X is a real number greater than 6 to 14, preferably from 7 to 10 andmost preferably from 8 to 9. In a preferred embodiment, the inventioncomprises, a cellular hematology control kit for glycated hemoglobincontained in red blood cells comprising a) red blood cells containingfrom about 4 to about 6 percent by weight of S-Alc glycated hemoglobinbased on the total amount of hemoglobin in the red blood cells; b) redblood cells containing from about 7 to about 9 percent by weight ofS-Alc glycated hemoglobin, or S-Alc glycated hemoglobin and acetylatedhemoglobin, based on the total amount of hemoglobin in the red bloodcells and one or more cis di-ahls; and c) red blood cells containingfrom about 12 to about 14 percent by weight of S-Alc glycatedhemoglobin, or S-Alc glycated hemoglobin and acetylated hemoglobin,based on the total amount of hemoglobin in the red blood cells and oneor more cis di-ahls. In another preferred embodiment, the inventioncomprises a cellular hematology control kit for glycated hemoglobincontained in red blood cells comprising a) red blood cells containingfrom about 4 to about 6 percent by weight of S-Alc glycated hemoglobinbased on the total amount of hemoglobin in the red blood cells; and b)red blood cells containing from about 10 to about 12 percent by weightof S-Alc glycated hemoglobin, or S-Alc glycated hemoglobin andacetylated hemoglobin, based on the total amount of hemoglobin in thered blood cells and one or more cis di-ahls. The red blood cells aredisposed in a stabilized suspension, preferably an aqueous suspension.In one embodiment of the invention the controls may include the S-Alcglycated hemoglobin, or S-Alc glycated hemoglobin and acetylatedhemoglobin without the presence of the cis di-ahls

In another embodiment, the invention comprises a method for determiningthe accuracy and reproducibility of the operation of an analyticalinstrument capable of measuring the glycated hemoglobin levelscomprising

a) providing a cellular hematology control as described herein in aknown reference quantity:b) determining the glycated hemoglobin level in said control of a) withthe instrument; andc) comparing the glycated hemoglobin levels obtained in b) with theknown reference quantity.

In certain embodiments, the desired level of S-Alc hemoglobin, S-Alcglycated hemoglobin, or S-Alc glycated hemoglobin and acetylatedhemoglobin, is substantially stabilized through adjusting the pH of thecontrol to about 6 to about 8. In yet additional embodiments, thedesired level of S-Alc glycated hemoglobin, S-Alc glycated hemoglobin,or S-Alc glycated hemoglobin and acetylated hemoglobin, is substantiallymaintained through the addition of glucose to the control at an amountof from about 0.001 to about 4 percent by weight. The term substantiallystabilized encompasses situations where the S-Alc hemoglobin level ofthe control varies by no more that about 1 to 2 percent, and preferably,varies by no more than about 1 percent over time. An additionalembodiment of the present invention includes a method for determiningthe accuracy of an analytical instrument capable of measuring S-Alcglycated hemoglobin levels comprising the steps of a) providing acellular S-Alc hemoglobin control of the present invention in a knownreference quantity (e.g. an S-Alc glycated hemoglobin cellularstandard); b) determining the level of S-Alc glycated hemoglobin in thecontrol of step a with the instrument; and c) comparing the S-Alcglycated hemoglobin level obtained from step b with the known referencequantity; wherein the comparing indicates the accuracy of saidhematology instrument. It will be desirable to obtain a value in step cthat is within about 1 to about 5 percent of the value of the knownreference quantity. In certain embodiments, the Level 1 cellularglycated S-Alc hemoglobin, S-Alc glycated hemoglobin, or S-Alc glycatedhemoglobin and acetylated hemoglobin, controls of the present inventionwill serve as control or reference standard provided in a knownreference quantity of step b, for the normal range of S-Alc glycatedhemoglobin levels (S-Alc glycated hemoglobin levels less than or equalto about 6 percent) for use in a variety of diagnostic equipment,including in analytical instrument capable of measuring S-Alc glycatedhemoglobin levels. In the compositions and controls of the invention, itis preferable that the concentration of S-Alc glycated hemoglobin, orS-Alc glycated hemoglobin, or S-Alc glycated hemoglobin and acetylatedhemoglobin, measured by high pressure liquid chromatography, immunoassayand by boronate affinity methods are consistent and are preferablywithin a range of about 2 percent and more preferably within a range ofabout 1 percent. Within a range of 2 percent means that the readings ofthe various analytical tests are within a range of 2 percent, forexample 9.1, 10.0 and 10.9. Although the three methods measure differentcomponents of the red blood cells, the software that controls apparatusused to perform the analysis can be adjusted to facilitate consistentresults between the two methods.

The control composition is prepared and analyzed by the same standardmethod as test samples which may be tested in batch quantities by theuse of a suitable cassette having apertures for receiving test vials.After preparation, the control composition and test samples are analyzedby detecting the presence of or counting the population number of eachsubject component type with a multi-parameter automated hematologyinstrument, which will preferably yield a visual display of the data. Inone embodiment the control of the present invention is provided incombination with a peripheral device, such as a device for trackingsamples and associating them with particular data, e.g., a bar-codescanner system, an MD system or otherwise. The control may also beprovided in combination with a slide preparation kit, stain ordye-resistant labels, lytic reagents (e.g., containing a quaternaryammonium salt), blood diluents, or other like components used in aclinical laboratory setting.

The skilled artisan will appreciate that a number of the steps andingredients have been disclosed by way of example, but that any of anumber of alternative steps or ingredients at the suggested or differentparameter or concentration, may be suitably substituted. Though theingredients or steps have been, in certain instances, described byreference to a particular function or result, it should be appreciatedthat such discussion is presented without intending to be bound bytheory. In some instances, the ingredient or step will perform adifferent or an additional function or achieve a different result, ormultiple other ingredients or steps may be substituted to perform suchfunction or achieve such result. Thus, there is no intention to be boundto the breadth of any specific illustrative step, parameter, ingredientor concentration, where it is apparent that others may be advantageouslybe employed in addition to or as a substitute.

The present invention is further illustrated by particular reference tothe following examples, it being understood that variations of the samemay be made while still remaining within the scope of the invention. Inthe examples that follow, as well as in accordance with the precedingteachings (to which the discussion in this paragraph also applies), itis expected that the resulting cellular components will be capable ofdetection by an automated hematology analyzer. The resulting sizes ofthe cellular components may be substantially the same as the startingcells, larger or smaller. When different, the cellular components mayrange from about one half to about twice the size of the original cells.In certain instances, it is also possible that hemoglobin may be removedfrom within the cell, with removals (when occurring) ranging from about0 percent to about 100 percent of the original hemoglobin (e.g., lessthan about 10 percent, less than about 20 percent, greater than about 70percent, greater than about 85 percent or otherwise).

SPECIFIC EMBODIMENTS OF THE INVENTION

The following experiments are included to illustrate the invention andare not intended to limit the scope of the claims. All parts andpercentages are by weight unless otherwise stated.

General Control Preparation Process

The Red Blood Cells (RBC cells) (stabilized RBC) (hemoglobin (HGB)concentration=12 g/dL, RBC concentration=4×10⁶/μL and S-Alc=5-6 percent)in stabilizing solution are contacted with carbon monoxide (CO) bybubbling CO through the dispersion of red blood cells. The CO-treatedRBC cells are then washed by adding the glycating solution of mannose orglucose and purine compound, 3 percent bovine serum albumin BSA in aglycating medium of phosphate buffer containing phosphate and variousantimicrobial agents) as generally described in Table 1, and then themixture is centrifuged for 15 minutes at 1500 rpm. The glycatingsolution is then removed by aspirating the supernatant. The washingprocess is repeated 3 times. The volume of the sample is adjusted suchthat the hemoglobin concentration is maintained as 11-12 g/dL.

The sample is glycated in a water bath at 37° C. for about 24 hours,glycation step. The hemoglobin concentration is measured at thebeginning (0 hr) and at the end (about 22 hours). The sample is allowedto cool to room temperature (RT) for the next step, deglycation.

The glycated samples are washed three times with a diluent solutioncontaining 3 percent bovine serum albumin (BSA) and no mannose, glucoseor purine compound. A typical washing procedure includes diluting thesample with diluent, mixing thoroughly and then centrifuging for 15minutes at 1500 rpm. The supernatant is removed by aspiration. The cellto the diluent ratio is maintained at 1:5. The sample is then deglycatedat 30° C. for about 24 hours (glucose glycating agent) or at roomtemperature where the glycating agent is mannose, de-glycation step.

The deglycated samples are then fixed with 0.005 percent glutaraldehydein a solution of phosphate buffer (pH 7 with a composition described inthe Table 3) at room temperature for 24 hours. The glutaraldehyde amountis calculated based on the RBC concentration of the sample. An equalvolume fixing diluent, containing 3 percent BSA and no glucose, isprepared with the calculated amount of the glutaraldehyde. The sample,once fixed with glutaraldehyde, is washed in diluent containing acis-diahl, the final storing diluent, (detailed composition is providedin Table 4), in order to remove the fixative. The sample is stored at 6°C. for long term storage.

TABLE 4 diluent Ingredient mg % mM % EDTA (Na₂) 704 18.09 0.704 MgGluconate 392 9.45 0.392 Na₂HPO₄ 268 18.87 0.268 PEG-20k 700 0.35 0.700Inosine 425 15.9 0.425 Glucose 6000 333.3 6.000 NaOH 80 20 0.080Methylparaben 40 2.6 0.040 Neomycin SO₄ 40 0.44 0.040 Chloramphenicol 150.46 0.15 Cis di-ahl 200, 300 2, 3 or 600 or 6

The stabilized solutions in the following examples are tested for S-Alcglycated hemoglobin levels. The S-Alc hemoglobin concentrations aremeasured by HPLC, Immunoassay and Boronate Affinity methods. The HPLCmeasurement is performed utilizing a Alc 2.2 Plus or G8 Analyzeravailable from TOSOH Bioscience utilizing manufacturer recommendedprocedures. The boronate affinity measurement is performed utilizing aCholestech GDX Analyzer with Cholestech GDX Alc Test Cartridges,available from Cholestech, and Cholestech recommended procedures. Theimmunoassay testing is performed utilizing a DCA Vantage analyzer,available from Siemens and Siemens recommended procedures.

Examples 1 to 16

Red blood cells are processed as described above adjusting the glucoseand catalyst concentration. The final stabilized product contained 3%percent sorbitol. The S-Alc concentrations are measured by HPLC,Immunoassay and Boronate Affinity. The results are compiled in Table 5.

TABLE 5 Condition (temp. = 37° C.) Final S-A1c cocn. (%) catalyst Immu-Example Glucose catalyst conc. HPLC noassay Affinity 1 6% adenosine0.50% 10.5 8.6 — 2 inosine 0.50% 10.9 10.5 8.6 3 6% inosine 0.50% 11.710.1 12.6 4 adenosine 0.50% 11.8 10.6 — 5 6% no catalyst 0.00% 9.2 8.69.9 6 adenosine 0.40% 9.4 8.9 10.4 7 inosine 0.20% 9.8 9.3 8.5 8 0.40%10.3 8.6 9.1 9 0.50% 9.7 9.4 9.4 10 6% inosine 0.40% 9.8 9.3 13.3 11 6%inosine 0.40% 10.4 9.5 10.4 12 5% inosine 0.30% 9.8 8.6 8 13 inosine0.40% 9.8 8.4 8 14 6% inosine 0.30% 9.9 9.2 9.7 15 inosine 0.40% 10 8.910.2 16 6% inosine 0.40% 10.4 9.3 9.7

Table 5 illustrates the impact of sorbitol on the consistency of thestandard analytical methods.

Examples 17 to 34

Several samples are prepared using levels of 3 and 6 percent of sorbitolin the stabilized final aqueous suspension. In the preparation of thesamples the level and purine catalyst are varied. The glucoseconcentration in the glycation step is 6 percent and the purine catalystlevel in 0.5 percent by weight, except where noted in the glycationsuspension. The final aqueous suspensions are tested for S-Alc glycatedhemoglobin concentration by boron affinity, high pressure liquidchromatography and immunoassay techniques. The results are compiled inTable 6. Level 1 as used in Table 6 means the diluent as described inTable 4.

TABLE 6 S-A1c Catalyst/ S-A1c Immuno- S-A1c Example ConcentrationDiluent Affinity assay HPLC 17 none Level 1 26.8 9.6 10 18 none Level1 + 10.8 9.2 9.5 Sorbitol 6% 19 adenosine (0.5) Level 1 20.5 8.7 11 20adenosine (0.5) Level 1 + 8.6 10.5 Sorbitol 6% 21 guanosine (0.5) Level1 16.5 9.0 10.9 22 guanosine (0.5) Level 1 + 11 9.6 10.7 Sorbitol 6% 23inosine (0.5) Level 1 17.8 10.8 11.1 24 inosine (0.5) Level 1 + 8.6 10.510.9 Sorbitol 6% 25 none Level 1 21.4 8.6 9.4 26 none Level 1 + 9.9 8.69.2 Sorbitol 3% 27 inosine (0.2) Level 1 16.9 9.6 9.8 28 inosine (0.2)Level 1 + 8.5 9.3 9.8 Sorbitol 3% 29 inosine (0.3) Level 1 16.1 9.6 10.330 inosine (0.3) Level 1 + 9.1 8.6 10.3 Sorbitol 3% 31 inosine (0.5)Level 1 15.9 9.7 9.7 32 inosine (0.5) Level 1 + 9.4 9.4 9.7 Sorbitol 3%33 adenosine (0.4) Level 1 20 9.3 9.6 34 adenosine (0.4) Level 1 + 10.48.9 9.4 Sorbitol 3%Table 6 illustrates the effect of sorbitol on the S-Alc valuesdetermined by affinity method.

Examples 35 to 37

Several samples are prepared as described hereinbefore using 2 percentof sorbitol in the stabilized final aqueous suspension. The mannoseconcentration in the glycation step is 6 percent. The final aqueoussuspensions are tested for S-Alc glycated hemoglobin concentration byboron affinity, high pressure liquid chromatography and immunoassaytechniques. The results are compiled in Table 7.

TABLE 7 S-A1c conc. of the final product Tosoh 2.2 Tosoh G8 GDXCholestech DCA Vantage Example (HPLC) (HPLC) (Affinity) (Immunoassay) 3511.7 11.1 10.2 10.9 36 No data 11.2 11.2 10.5 37 12.1 11.7 11.2 10.7

Table 7 illustrates the effect of sorbitol on the S-Alc valuesdetermined by affinity method and shows that the results of all threemethods are consistent.

General Control Preparation Process

Examples 38 to 78

The Red Blood Cells (RBC cells) (stabilized RBC) (hemoglobin (HGB)concentration=12 g/dL, RBC concentration=4×10⁶/μL and S-Alc=5-6 percent)in stabilizing solution are contacted with carbon monoxide (CO) bybubbling CO through the dispersion of red blood cells. The CO-treatedRBC cells are then washed by adding the glycating solution of 6 percentglucose, 0.4 percent inosine, 3 percent bovine serum albumin BSA in aglycating medium of phosphate buffer containing phosphate and variousantimicrobial agents) as generally described in Table 1, and then themixture is centrifuged for 15 minutes at 1500 rpm. The glycatingsolution is then removed by aspirating the supernatant. The washingprocess is repeated 3 times. The volume of the sample is adjusted suchthat the hemoglobin concentration is maintained as 11-12 g/dL.

The sample is glycated in a water bath at 37° C. for about 24 hours. Thehemoglobin concentration is measured at the beginning (0 hr) and at theend (about 22 hours). The sample is allowed to cool to room temperature(RT) for the next step, deglycation. The glycated samples are washedthree times with a diluent solution containing 3 percent bovine serumalbumin (BSA) and no glucose. A typical washing procedure includesdiluting the sample with diluent, mixing thoroughly and thencentrifuging for 15 minutes at 1500 rpm. The supernatant is removed byaspiration. The cell to the diluent ratio is maintained at 1:5. Thesample is then deglycated at 30° C. for about 24 hours, de-glycationstep.

The deglycated samples are then fixed with 0.002 percent glutaraldehydein a solution of phosphate buffer (pH 7 with a composition described inthe Table 3) at room temperature for 24 hours. The glutaraldehyde amountis calculated based on the RBC concentration of the sample. An equalvolume fixing diluent, containing 3 percent BSA and no glucose, isprepared with the calculated amount of the glutaraldehyde. The sample,once fixed with glutaraldehyde, is washed in diluent-1, the finalstoring diluent, detailed composition is provided in Table 4), in orderto remove the fixative. The sample is stored at 6° C. for long termstorage.

The stabilized solutions in the following examples are tested for S-Alcglycated hemoglobin levels. The S-Alc hemoglobin concentrations aremeasured by HPLC, Immunoassay and Boronate Affinity methods. The HPLCmeasurement is performed utilizing a Alc 2.2 Plus Analyzer availablefrom TOSOH Bioscience utilizing manufacturer recommended procedures. Theboronate affinity measurement is performed utilizing a Cholestech GDXAnalyzer with Cholestech GDX Alc Test Cartridges, available fromCholestech, and Cholestech recommended procedures. The immunoassaytesting is performed utilizing a DCA Vantage analyzer, available fromSiemens and Siemens recommended procedures.

Examples 38 to 50

Red blood cells are processed as described above with various purinebased compounds at 0.4 and 0.5 percent by weight of the glycationsuspension. The S-Alc glycated hemoglobin concentrations are measuredprior to glycation, after glycation, after deglycation and in the finalstabilized solution (final) by Tosoh Alc 2.2 Plus (1-HPLC). The resultsare compiled in Table 8.

TABLE 8 Catalyst S-A1c conc. (%) Ex- Conc. ini- Post- post- ample Name(%) tial glycation deglycation final 38 no catalyst 0 6 10 10.8 11.1 39Inosine 0.5 6 11.5 12.3 11.8 40 adenosine* 0.5 6 11.1 12.1 11.7 41guanosine* 0.5 6 9.8 10.9 10.7 42 no catalyst 0 6.2 8.6 9.7 9.4 43inosine 0.4 6.2 10.6 11.3 10.3 44 adenosine 0.4 6.2 10.6 11.2 9.6 45 nocatalyst 0 5.5 8.4 9.4 9.2 46 inosine 0.4 5.5 10.1 11.1 10.3 47adenosine 0.4 5.5 10 11 9.8 48 xanthosine 0.4 5.5 9.7 10.7 9.6 49inosine 0.4 5.5 10.4 11.6 Not pro- 50 2-amino- 0.4 5.5 9.7 11.3 cessedadenosine* *was not completely soluble at that concentrationTable 8 illustrates effect of purine compounds on the S-Alc synthesisrate.

Examples 51 to 53

Red blood cells are processed as described above using other purinecompounds and the S-Alc hemoglobin concentrations are measured by HPLCmethods. The results are compiled in Table 9.

TABLE Catalyst S-A1c conc. (%) Example Name Conc. initial Post-glycation51 No catalyst 0 6.2 8.5 52 AMP 0.5% 6.2 9 53 Cyclic AMP 0.5% 6.2 8.8Table 9 illustrates that presence of one phosphate linkage reduces thecatalytic efficiency of the purine derivative.

Examples 54 to 69

Red blood cells are processed as described hereinbefore at varioustemperatures, 22° C., 30° C. and 37° C. and samples are tested usingTosoh Alc 2.2 Plus (HPLC) test at various times for S-Alc to determinethe best temperature to result in the formation of red blood cellshaving 9 percent by weight of greater S-Alc glycated hemoglobin based onthe weight of the hemoglobin. The concentration of glucose in theglycation medium is 6 percent by weight of the glycation medium. Theresults are compiled in Table 10.

TABLE 10 S-A1c conc. at different times of glycation Example Temp. A1c 0hr 18 hr 24 hr 48 hr 54 22° C. L-A1c 4.2 19.6 20.1 19.6 55 S-A1c 6.6 6.56.5 6.6 56 30° C. L-A1c 4.2 19.3 19.3 18.2 57 S-A1c 6.6 6.6 6.6 7.1 5837° C. L-A1c 4.2 17.4 16.6 12 59 S-A1c 6.6 7.1 7.4 9 60 30° C. L-A1c 5.113.6 12.4 11.3 61 S-A1c 6.6 6.7 6.8 7.7 62 37° C. L-A1c 5.1 11.6 11.2 63S-A1c 6.6 8.5 8.9 64 22° C. L-A1c 5.2 21.4 21.6 16 65 S-A1c 6.6 6.5 6.56.7 66 30° C. L-A1c 5.2 16.8 14.7 11.8 67 S-A1c 6.6 6.6 6.7 7.6 68 37°C. L-A1c 5.2 12.5 12.8 69 S-A1c 6.6 8.2 8.6

Table 10 illustrates the effect of temperature on S-Alc synthesis.

Examples 70 to 74

Red blood cells are processed as described above and the S-Alcconcentrations are measured by the HPLC (Tosoh Alc 2.2 Plus) method. TheBSA concentration is varied. The results are compiled in Table 11.

TABLE 11 Example 5% Glucose + 0.5% inosine 70 BSA Conc. 3% 5% 10%  71L-A1c (0 hr) 2.2 2.2 2.2 72 L-A1c (22 hr) 8.9 8.9 8.9 73 S-A1c (0 hr)6.2 6.2 6.2 74 S-A1c (22 hr) 10.7  10.6  10.7 

Table 11 illustrates that concentration of BSA has no effect onglycation rate.

Examples 75 to 88

Red blood cells are processed as described above and the S-Alchemoglobin concentrations are measured by HPLC at various times todetermine the amount of time needed to achieve a concentration of S-Alcglycated hemoglobin using 6 percent glucose and 0.4 percent inosine at37° C. The results are compiled in Table 12.

TABLE 12 Final Ex- HGB A1c conc. (%) during glycation A1c ample (g/dL) 020 21 23 conc. (%) S-A1c 75 9.9 6.4 9.5 9.3 9.6 10.7 76 9.6 6.3 9.1 9.19.6 10.4 77 10.4 5.8 9 8.8 9.2 10 78 10.2 6.5 9.4 9.4 9.8 10.4 79 9.86.5 9.4 9.5 9.8 10.4 L-A1c 80 9.9 5.1 9.4 9.3 9.6 5.9 81 9.6 5.1 9.3 9.29.4 5.9 82 10.4 6 9.5 9.6 9.7 5.1 83 10.2 5.3 9.1 9.2 9.4 5.5 84 9.8 5.39.2 9.1 9.4 5.5 S-A1c 85 10.7 6.6 9.9 9.9 10 10.3 86 11 6.6 9.8 9.9 10.210.4 L-A1c 87 10.7 10 8.7 8.6 8.9 4.2 88 11 10 8.8 8.7 9 4.5Table 12 illustrates the increase in concentration of S-Alc and L-Alcformation with time.

Experimental Process Examples 89-127

The Red Blood Cells (RBC cells) (stabilized RBC) (hemoglobin (HGB)concentration=12 g/dL, RBC concentration=4×10⁶/μL and S-Alc=5-6 percent)in stabilizing solution are contacted with carbon monoxide (CO) bybubbling CO through the dispersion of red blood cells. The CO-treatedRBC cells are then washed by adding the glycating solution of 5 percentglucose, 0.25 percent acetyl salicylic acid (ASA), 3 percent bovineserum albumin BSA in a glycating medium of phosphate buffer containingphosphate and various antimicrobial agents) as generally described inTable 1, and then the mixture is centrifuged for 15 minutes at 1500 rpm.The glycating solution is then removed by aspirating the supernatant.The washing process is repeated 3 times. The volume of the sample isadjusted such that the hemoglobin concentration is maintained as 11-12g/dL. The sample is glycated in a water bath at 37° C. for 20 to 22hours. The hemoglobin concentration is measured at the beginning (0 hr)and at the end (about 22 hours). The sample is allowed to cool to roomtemperature (RT) for the next step, deglycation. The glycated samplesare washed three times with a diluent solution containing 3 percentbovine serum albumin (BSA) and no glucose. A typical washing procedureincludes diluting the sample with diluent, mixing thoroughly and thencentrifuging for 15 minutes at 1500 rpm. The supernatant is removed byaspiration. The cell to the diluent ratio is maintained at 1:5. Thesample is then deglycated at 30° C. for about 24 hours.

The resulting samples are then fixed with 0.002 percent glutaraldehydein a solution of phosphate buffer (pH 7 with a composition described inthe Table 3) at room temperature for 24 hours. The glutaraldehyde amountis calculated based on the RBC concentration of the sample. An equalvolume fixing diluent, containing 3 percent BSA and no glucose, isprepared with the calculated amount of the glutaraldehyde. The sample,once fixed with glutaraldehyde, is washed in diluent-1, the finalstoring diluent, detailed composition is provided in Table 4), in orderto remove the fixative. The sample was stored at 6° C. for long termstorage.

Examples 89 to 94

Red blood cells are processed as described above and the S-Alchemoglobin concentrations are measured by HPLC and Boronate Affinitymethods. The HPLC measurement is performed utilizing a Alc 2.2 PlusAnalyzer available from TOSOH Bioscience utilizing manufacturerrecommended procedures. The boronate affinity measurement is performedutilizing a Cholestech GDX Analyzer with Cholestech GDX Alc TestCartridges, available from Cholestech, and Cholestech recommendedprocedures. The results are compiled in Table 13.

TABLE 13 Glycation Condition S-A1c conc. (%) Example Glucose Temp. TimeHPLC Boronate affinity 89 6% 37° C. 96 hr 16.9 35.7 90 6% 37° C. 72 hr11.5 28.7 91 6% 37° C. 48 hr 11.4 23 92 6% 37° C. 22 hr 9.5 15 93 5% 37°C. 22 hr 9 13.8 94 4% 37° C. 22 hr 8.3 9.8

Examples 85 to 103

Red blood cells are processed as described above and the S-Alchemoglobin concentrations are measured by HPLC and Boronate Affinitymethods. The concentrations of glucose and acetyl salicylic acid and theincubation times are varied. The results are compiled in Table 14.

TABLE 14 Conditions of incubation S-A1c (%) Glucose ASA Time (afterincubation at 37° C.) Example (%) (%) (hr) HPLC Boronate Affinity 95 50.2 22.0 9.8 9.1 96 5 0.25 22.0 10.2 10.4 97 5 0.3 15 10.2 11.3 98 5 0.322.0 10.6 9.8 99 5 0.4 5.5 9.9 6.7 100 6 0.2 15 9.4 10.3 101 6 0.3 1510.3 12.5 102 10 0.2 22.0 10.8 13.6 103 10 0.4 5.5 9.3 6.4

Table 14 illustrates that the right combination of the concentrations ofglucose and the acetylating agent, ASA, is important to maintain anaccelerated incubation process and to obtain an equal value of S-Alchemoglobin concentration measured as measured by HPLC and boron affinitymethods. In these examples, the S-Alc hemoglobin concentration of asample, obtained by this procedure, determined by HPLC, is theconcentration of the combined glycated hemoglobin and acetylatedhemoglobin. Therefore, the HPLC reported value is referred as S-Alc peakarea. On the other hand, S-Alc concentration determined by the boronateaffinity method is solely due to the glycated hemoglobin. Thus, theboronate affinity method reported value is referred as S-Alcconcentration. In general, the S-Alc concentrations, reported bydifferent methods, are referred as S-Alc value. The higher concentrationof glucose during incubation, at a given ASA concentration, increasesthe rate of the S-Alc synthesis and the values of S-Alc concentrationreported by both of the HPLC and boronate affinity methods. The S-Alcvalues measured by the boronate affinity method are increased many foldbecause of the increase in concentrations of the minor components. Thehigher concentration of ASA, on the other hand, increases the S-Alc peakarea in HPLC and the rate at which the increase occurs. However, theS-Alc value reported by the boronate affinity method is found todecrease with higher ASA at a constant glucose concentration. Higher (≧6percent) glucose concentrations caused hemolysis. Therefore, 5-6 percentis the optimum glucose concentration. Incubation with 0.3 percent ASAprovides a sample with the most reproducible value of S-Alcconcentration. (Acetylation produces many transiently stableacetylated-hemoglobin species which may contribute to the fluctuatingS-Alc concentration values.)

Examples 104 to 117

Red blood cells are processed as described above and the S-Alcconcentrations are measured by HPLC and Boronate Affinity. Theconcentration of acetyl salicylic acid is varied. The results arecompiled in Table 15.

TABLE 15 Conditions of incubation S-A1c (%) Example Glucose (%) ASA (%)HPLC Affinity 104 5 0.2 10.2 8.6 105 5 0.2 10.2 13.8 106 5 0.2 11.1 12.1107 5 0.2 10.4 8.6 108 5 0.3 10.8 10.7 109 5 0.3 10.6 10.6 110 5 0.310.9 12.1 111 5 0.3 11.7 11.3 112 5 0.4 11.4 11.8 113 6 0.2 10.4 na 1146 0.3 11.1 12.2 115 7 0.2 10.7 14.5 117 7 0.3 11.5 11.4 118 7 0.4 11.97.1The deglycation of RBC erythrocytes with 5 to 6 percent glucose combinedwith 0.2 to 0.3 percent ASA at 37° C. for 24 hr results in an increaseof S-Alc concentration values from 5 to 6 percent range to the 9 to 10percent range. The preferred condition are 5 percent glucose with 0.3percent ASA.

Examples 119 to 121

Red blood cells are processed as described above and the S-Alcconcentrations are measured by HPLC and Boronate Affinity methods. Theratio of glucose to hemoglobin is varied. In order to obtain a desiredtarget concentration of S-Alc in a range of 10□ to 11 percent, thehemoglobin concentration (HGB) is maintained in the 11-12 g/dL range.The results are compiled in Table 16.

TABLE 16 1 mol of glucose + 0.04 Sample mol of ASA 37° C. HGB conc. HGBRBC after 15 hr Example (g/dL) (×10⁻³ mol) (×10¹² mol) L-A1c S-A1c 1198.9 4.96 9.9 11.5 10.8 120 12.6 7.01 13.74 11.1 10.1 121 16.7 9.32 18.1310.7 9.6

Examples 122 to 127

Red blood cells are processed as described above and the S-Alchemoglobin concentrations are measured by HPLC and Boronate Affinity.The deglycation is performed with 5 percent glucose, 0.25 percent byweight acetyl salicylic acid at 37° C. The results are compiled in Table17.

TABLE 17 Reaction time (hr) 0 15 20 24 30 S-A1c Example 124 6.3 8.6 8.79.3 9.6 Example 125 6.2 8.3 8.5 9.1 9.4 L-A1c Example 126 6.3 7.1 7 7.16.9 Example 127 6.3 7.2 7.1 7.1 6.9The reaction time is determined based on the desired target (postglycation stage) of S-Alc concentration. In order to obtain about 9-10percent S-Alc concentration of the final product, the glycation isstopped at about 20-22 hours when the S-Alc concentration is about 9-10percent.

It will be appreciated that concentrates or dilutions of the amountsrecited herein may be employed. In general, the relative proportions ofthe ingredients recited will remain the same. Thus, by way of example,if the teachings call for 30 parts by weight of a Component A, and 10parts by weight of a Component B, the skilled artisan will recognizethat such teachings also constitute a teaching of the use of Component Aand Component B in a relative ratio of 3:1.

It will be appreciated that the above is by way of illustration only.Other ingredients may be employed in any of the compositions disclosedherein, as desired, to achieve the desired resulting characteristics.Examples of other ingredients that may be employed include antibiotics,anesthetics, antihistamines, preservatives, surfactants, antioxidants,unconjugated bile acids, mold inhibitors, nucleic acids, pH adjusters,osmolarity adjusters, or any combination thereof. Specific examples ofingredients that may be employed include one or more of sodium fluoride,a paraben (e.g., propyl), sulfasalazine, sodium phosphate, potassiumphosphate, sodium citrate, citric acid, sodium chloride, bovine serumalbumin, sodium hydroxide, lipoprotein, Proclin, adenine, mannose,dextrose, lactose, penicillin, tetracycline, promethazine, a purine(e.g., adenine), inosine, kanamycin sulfate, cyclohexamide, deoxycholicacid, colistimethate sodium, trisodium citrate dehydrate,5-Fluorouracil, or any combination thereof.

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

1. A composition comprising red blood cells in a suspension medium andone or more cis di-ahls; wherein more than 6 percent by weight of thehemoglobin in the red blood cells is S-Alc glycated.
 2. A compositionaccording to claim 1 wherein the suspension medium is an aqueoussuspension medium.
 3. A composition according to claim 1 which furthercomprises from about 0.3 to about 0.6 percent by weight based on theweight of glucose based on the weight of the composition.
 4. Acomposition according to claim 1 wherein the one or more cis di-ahlscomprise one or more compounds having two active hydrogen containingfunctional groups on adjacent carbon atoms.
 5. A composition accordingto claim 4 wherein the active hydrogen containing functional groups areselected from the group of hydroxyl, amino, thiol and carboxylatefunctional groups.
 6. A composition according to claim 5 wherein theactive hydrogen containing functional groups are hydroxyl.
 7. Acomposition according to claim 1 wherein the one or more cis di-ahls arepresent in the composition in an amount of about 1 to about 6 percent byweight based on the weight of the composition.
 8. A compositionaccording to claim 1 wherein the source of the red blood cells is anon-diabetic human.
 9. A composition according to claim 1 wherein theconcentration of S-Alc glycated hemoglobin measured by high pressureliquid chromatography, immunoassay and boronate affinity methods areconsistent.
 10. A composition according to claim 1 wherein theconcentration of S-Alc glycated hemoglobin measured by high pressureliquid chromatography, immunoassay and boronate affinity methods arewithin a range of units of about 2 percent.
 11. A method comprisingcontacting red blood cells in a suspension medium having a concentrationof S-Alc glycated hemoglobin of greater than 6 percent by weight of thehemoglobin in the red blood cells with a sufficient amount of one ormore cis di-ahls such that the concentration of S-Alc glycatedhemoglobin in resulting composition as measured by measured by highpressure liquid chromatography, immunoassay and boronate affinitymethods is consistent.
 12. A method according to claim 11 wherein thesuspension medium is an aqueous suspension medium.
 13. A methodaccording to claim 11 wherein the amount of the one or more cis di-ahlscontacted with the red blood cells in the suspension medium is about 1to about 6 percent by weight based on the weight of the suspensionmedium.
 14. A method according to claim 11 wherein the one or more cisdi-ahl comprise one or more compounds having two active hydrogencontaining functional groups on adjacent carbon atoms
 15. A methodaccording to claim 11 wherein the suspension medium contains from about0.3 to about 0.6 percent by weight of glucose based on the weight of thesuspension medium.
 16. A cellular hematology control for glycatedhemoglobin contained in red blood cells comprising intact red bloodcells having a predetermined level of greater than about 6 percent byweight of S-Alc glycated hemoglobin and one or more cis di-ahls in astabilized suspension
 17. A cellular hematology control according toclaim 16 wherein the one or more cis di-ahls are present in an amount ofabout 1 to about 6 percent by weight based on the weight of thestabilized suspension medium.
 18. A cellular hematology control forglycated hemoglobin contained in red blood cells comprising a) red bloodcells in a stabilized aqueous medium wherein the hemoglobin containsfrom about 4 to about 6 percent by weight of S-Alc glycated hemoglobinbased on the weight: of the hemoglobin; b) red blood cells in astabilized aqueous medium wherein the hemoglobin contains greater thanabout 6 and less about X percent by weight of S-Alc glycated hemoglobinbased on the weight of the hemoglobin and about 1 to about 6 percent byweight of one or more cis di-ahls based on the weight of the stabilizedaqueous medium; and c) red blood cells in a stabilized aqueous mediumwherein the hemoglobin contains greater than about X to about 14 percentby weight of S-Alc glycated hemoglobin hemoglobin based on the weight ofthe hemoglobin and about 1 to about 6 percent by weight of one or morecis di-ahls based on the weight of the stabilized aqueous medium;wherein X is between about 7 and
 14. 19. A cellular hematology controlfor glycated hemoglobin contained in red blood cells according to claim16 wherein the source of the red blood cells is a non-diabetic human.20. A method for determining the accuracy and reproducibility of theoperation of an analytical instrument capable of measuring the glycatedhemoglobin levels comprising a) providing a cellular hematology controlaccording to claim 16 in a known reference quantity: b) determining theglycated hemoglobin level in said control of a) with the instrument; andc) comparing the glycated hemoglobin levels obtained in b) with theknown reference quantity.
 21. A composition comprising red blood cellshaving a portion of the hemoglobin as acetylated hemoglobin and aportion of hemoglobin as S-Alc glycated hemoglobin, wherein the redblood cells are glycated on the terminal valine amino acid of the Alphaor Beta chain, with the proviso that more than 6 percent by weight ofthe hemoglobin in the red blood cells are acetylated or glycated on theterminal valine amino acid of the Alpha or Beta chain.
 22. A methodcomprising contacting red blood cells in a suspension medium containingglucose and a compound containing a purine ring structure having aribose sugar ligand attached the five membered ring of the purine ringstructure at about 35° C. to about 40° C. under conditions such that theconcentration of S-Alc glycated hemoglobin is increased to greater thanabout 6 percent by weight of the hemoglobin in the red blood cells.